AADvac1, an Active Immunotherapy for Alzheimer's Disease and Non Alzheimer Tauopathies: An Overview of Preclinical and Clinical Development.

Neurofibrillary tau protein pathology is closely associated with the progression and phenotype of cognitive decline in Alzheimer's disease and other tauopathies, and a high-priority target for disease-modifying therapies. Herein, we provide an overview of the development of AADvac1, an active immunotherapy against tau pathology, and tau epitopes that are potential targets for immunotherapy. The vaccine leads to the production of antibodies that target conformational epitopes in the microtubule-binding region of tau, with the aim to prevent tau aggregation and spreading of pathology, and promote tau clearance. The therapeutic potential of the vaccine was evaluated in transgenic rats and mice expressing truncated, non mutant tau protein, which faithfully replicate of human tau pathology. Treatment with AADvac1 resulted in reduction of neurofibrillary pathology and insoluble tau in their brains, and amelioration of their deleterious phenotype. The vaccine was highly immunogenic in humans, inducing production of IgG antibodies against the tau peptide in 29/30 treated elderly patients with mild-to-moderate Alzheimer's. These antibodies were able to recognise insoluble tau proteins in Alzheimer patients' brains. Treatment with AADvac1 proved to be remarkably safe, with injection site reactions being the only adverse event tied to treatment. AADvac1 is currently being investigated in a phase 2 study in Alzheimer's disease, and a phase 1 study in non-fluent primary progressive aphasia, a neurodegenerative disorder with a high tau pathology component.

Alternative hypotheses related to Alzheimer's disease.

Alzheimer's disease represents the most common form of dementia and belongs to the group of neurodegenerative disorders characterized by progressive loss of neurons in the central nervous system. In the pathogenesis of Alzheimer's disease several etiologic and pathogenic factors exist, which lead to the dysfunction of neurotransmitter systems and consequent cognitive decline. Last three decades have delivered a crucial progress leading to better understanding of Alzheimer's disease, however, the exact mechanisms of pathology remain unclear. In this review, we summarize some hypotheses such as amyloid and tau hypotheses, inflammatory processes, prion-like hypothesis, the hypothesis of oxidative stress, vascular and cholesterol hypothesis, the hypothesis of metal accumulation in the brain, cell cycle hypothesis, the hypothesis of impaired insulin signalization and another, which were proposed to explain the pathogenesis of this severe disorder (Ref. 115).

How can viruses influence the neuroinflammation and neurodegeneration in the aged human brain.

Age is one of the key risk factors of several human neurodegenerative disorders such as Alzheimer΄s disease and Parkinson΄s disease. During aging the immune system of the brain undergoes multiple structural and functional changes. The major immune cells of the brain - microglia and astrocytes - significantly change their morphology and functional state during aging. Similarly, the blood brain barrier (BBB), that is considered to be the iron curtain protecting the brain parenchyma against invasion of the pathogens, can be influenced by aging. This state of altered brain immunity may lead to the increased brain vulnerability to viral infections, primoinfection as well as reactivation. We hypothesize that impairment of the brain immunity and BBB integrity can create the optimal condition for viral infection that can further amplify the neuroinflammation mediated by glial cells and neurodegeneration induced and driven by disease modified proteins.

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ).

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.

Mesenchymal stem cells rescue the Alzheimer's disease cell model from cell death induced by misfolded truncated tau.

We have developed a stably transfected human cell model for Alzheimer's disease with doxycycline-inducible expression of human misfolded truncated tau protein (AT tau). We have showed that AT tau reduced the metabolic activity of the AT tau cells, slowed down cell proliferation, and induced caspase-3-independent apoptosis-like programmed cell death, tauoptosis. The aim of this study was to test the possible capability of rat mesenchymal stem cells (MSCs) to interfere with AT tau protein-induced cell death. AT tau cells after treatment with 10 µM all-trans retinoic acid were either co-cultivated with MSCs or supplemented with MSC secretome for 6 and 9 days. We found that both MSCs and MSC secretome promoted survival and increased the metabolic activity of the cells. Moreover stem cells induced cell differentiation and formation of neurites with numerous varicosities. Strikingly, treatment had no effect on tau expression suggesting that MSC induced self-protecting mechanism that prevented AT tau cells from tauoptosis. Our results showed that mesenchymal stem cells and their secretome are able to rescue the Alzheimer's disease cell model from cell death induced by misfolded truncated tau. We suggest that cell therapy may represent an alternative therapeutic avenue for treatment of human Alzheimer's disease and related tauopathies.

Risk and protective factors for sporadic Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of senile dementia. There are 24.3 million people suffering from this progressive neurodegenerative disorder worldwide. A century ago, AD was characterized with regard to the clinical manifestations and pathology for the first time. Up till now, there is a lack of full understanding of the underlying causes and molecular mechanisms leading to this progressive form of dementia. The majority of AD cases occur sporadically, what suggested that they could arise through interactions among various genetic and environmental factors. Current epidemiological investigations show that midlife hypertension, cardiovascular diseases, hypercholesterolemia, diabetes, obesity, inflammation, and viral infections can significantly contribute to the development and progression of AD, whereas active engagement in social, mental and physical activities may delay the onset of the disease. Apolipoprotein E (ApoE) is considered as the main genetic risk factor in the sporadic AD that is closely connected to lipid metabolism. Other genes involved in the disease pathways related to AD pathology in addition to cholesterol metabolism, neuroinflammation, amyloid and tau cascade, neuronal signalling, and plasticity are under investigation. In spite of the significant progress achieved, it is still not clear how genetic vulnerability and environmental exposures may contribute to the susceptibility of the disease. Therefore, understanding the role of disease-related risk factors for AD pathogenesis may help to identify specific modifiable risk factors that could provide possibility for the prevention of Alzheimer's dementia.

The tangled story of Alois Alzheimer.

In 1907, Bavarian psychiatrist Alois Alzheimer, who is considered to be a founding father of neuropathology, was first to describe the main neuropathologic characteristics of the peculiar disease in the brain of a woman showing progressive dementia when she was in her early 50s. Using a newly developed Bielschowsky's silver staining method, Alzheimer observed degenerating neurons with bundles of fibrils (neurofibrillary tangles) and miliary foci of silver-staining deposits scattered over the cortex (senile plaques). In 1910 Emil Kraepelin (Alois Alzheimer's superior) coined the term "Alzheimer's disease" to distinguish the presenile form of dementia from the more common senile variant. Alzheimer's findings were followed up, and soon a number of reports of similar cases appeared in the literature. During the time, both pathological hallmarks of Alzheimer's disease became the gold standard for post-mortem diagnosis of the disease. One hundred years later, dementia of Alzheimer's type is considered to be one of the most devastating illnesses of old age. Despite intensive research the cause of the disease still remains elusive (Fig. 2, Ref. 17).

The hunt for dying neurons: insight into the neuronal loss in Alzheimer's disease.

Neuronal loss is one of the major pathological hallmarks of neurodegenerative disorders including Alzheimer's disease (AD). Using rigorous quantitative methods, the distinct pattern of neuronal loss in pathological conditions such as neurodegeneration and in normal aging was clearly shown. Furthermore, the decrease of total neuronal numbers correlated in a considerable extent with the presence of neurofibrillary degeneration in the same brain regions. However, it appears that neurofibrillary tangles are not the only cause of reduction of neuronal populations, but also alternative triggers could induce neuronal death in this disease. Various inducers, most probably, activate different cell death pathways. Recently, apoptosis has been implicated as a possible mechanism for neuronal death. There is essentially no evidence of apoptosis in AD that would meet all criteria of its classical definition. Therefore it was suggested, that other modes of cell death could contribute to neuronal loss in AD and related disorders (Tab. 2, Ref. 70).

Neuroinflammation in Alzheimer's disease: protector or promoter?

Alzheimer's disease (AD) is an irreversible, progressive and degenerative disorder that destroys the higher structures of the brain. Prominent neuropathologic features of AD are senile plaques, neurofibrillary tangles, synaptic and neuronal loss. There is mounting evidence that chronic inflammatory processes play a fundamental role in the progression of neuropathological changes of AD. It has been shown, that there is a reciprocal relationship between the local inflammation and senile plaques (SPs) and neurofibrillary tangles (NFTs). The major players involved in the inflammatory process in AD are thought to be the microglia and the astrocytes. The process of the activation of glia is characteristized by upregulation or newly expression of a variety of molecules involved in inflammatory response including cytokines, various components of the complement cascade, acute phase reactants, proteases and protease inhibitors, and neurotoxic products. The importance of inflammation in the pathogenesis of AD was indirectly confirmed by epidemiological investigations that revealed a decreased incidence of AD in subjects using anti-inflammatory drugs, especially the non-steroidal anti-inflammatory drugs (NSAIDs). However clinical trials designed to inhibit inflammation have failed in the treatment of AD patients suggesting that anti-inflammatory agents have more protective than therapeutic effect. Despite the ongoing research the extent to which neuroinflammation contributes to disease pathogenesis is still not fully understood. Moreover it is also not clear whether the inflammation in AD brains represent a protective reaction to neurodegeneration or it is rather a destructive process that contributes to further loss of brain function. (Ref. 117).

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19-20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94-96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p < 0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi- and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p < 0.001) and lower total cell number (p < 0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

Antigenic relationship between five isolates of murine gammaherpesvirus analysed with monoclonal antibodies.

A panel of six monoclonal antibodies (mAbs) specific for murine gammaherpesvirus (MHV) was used for analysis of the antigenic relationship between five MHV-isolates (MHV 68, MHV 72, MHV 76, MHV 78, MHV S). Two mAbs raised against MHV 72 and four raised against MHV S were used in the study. Antibody-virus interactions were tested using immunochemical (ELISA, Western blot, immunofluorescence) and biological (virus-neutralization) assays. Immunoanalysis by ELISA showed a close antigenic relationship between the five viruses, nevertheless, some antigenic individuality of the isolate MHV S was observed. This isolate originated from a geographically distinct area in Czechia relative to the other four isolates from Slovakia. In Western blot analysis, antibodies to MHV 72 recognized viral antigens with the relative molecular mass about 116,000. Of four mAbs against MHV S, only two reacted with denatured viral antigen in Western blot and showed specificity for the 50-55,000 protein. These findings suggested that both isolates, besides of minor antigenic variability, could differ also in immunodominant proteins. Mabs to MHV S exhibited much stronger virus-neutralizing potency than mAbs to MHV 72, indicating thus that the 50-55,000 antigen might be more relevant for the infectivity of MHV-virus. Immunofluorescence with mAbs allowed specific localization of antigens in virus-infected VERO cells.