Matthias Gralle

Alzheimer's Disease-Like Pathology Induced by Amyloid-β Oligomers in Nonhuman Primates

Alzheimers disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-β (Aβ) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble Aβ oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, Aβ oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where Aβ oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with Aβ oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links Aβ oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.

Folding and Stability of the Extracellular Domain of the Human Amyloid Precursor Protein

The β-amyloid peptide (Aβ), the major component of the senile plaques found in the brains of Alzheimers disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by α- or β-secretases releases the extracellular soluble fragments sAPPα or sAPPβ, respectively. Despite the fact that sAPPα plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPPα695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513–3524). We now report studies on the folding and stabilities of sAPPα695 and sAPPα770. The combined use of intrinsic fluorescence, 4–4′-Dianilino-1,1′binaphthyl-5,5′-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPPα695 and sAPPα770 by GdnHCl and urea revealed multistep folding pathways for both sAPPα isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPPα. Furthermore, the relatively low stability of the native state of sAPPα suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.

Solution Studies and Structural Model of the Extracellular Domain of the Human Amyloid Precursor Protein

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimers disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.

The neuronal insulin receptor in its environment

Insulin is known mainly for its effects in peripheral tissues, such as the liver, skeletal muscles and adipose tissue, where the activation of the insulin receptor (IR) has both short‐term and long‐term effects. Insulin and the IR are also present in the brain, and since there is evidence that neuronal insulin signaling regulates synaptic plasticity and that it is impaired in disease, this pathway might be the key to protection or reversal of symptoms, especially in Alzheimers disease. However, there are controversies about the importance of the neuronal IR, partly because biophysical data on its activation and signaling are much less complete than for the peripheral IR. This review briefly summarizes the neuronal IR signaling in health and disease, and then focuses on known differences between the neuronal and peripheral IR with regard to alternative splicing and glycosylation, and lack of data with respect to phosphorylation and membrane subdomain localization. Particularities in the neuronal IR itself and its environment may have consequences for downstream signaling and impact synaptic plasticity. Furthermore, establishing the relative importance of insulin signaling through IR or through hybrids with its homolog, the insulin‐like growth factor 1 receptor, is crucial for evaluating the consequences of brain IR activation. An improved biophysical understanding of the neuronal IR may help predict the consequences of insulin‐targeted interventions.

Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimers disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Brain infusion of α-synuclein oligomers induces motor and non-motor Parkinson’s disease-like symptoms in mice

Abstract Parkinson’s disease (PD) is characterized by motor dysfunction, which is preceded by a number of non‐motor symptoms including olfactory deficits. Aggregation of &agr;‐synuclein (&agr;‐syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of &agr;–syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of &agr;‐syn oligomers (&agr;‐SYOs) in mice and evaluated motor and non‐motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that &agr;‐SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45 days post‐infusion (dpi). &agr;‐ SYO‐infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety‐like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of &agr;‐SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH‐positive cells was also seen in the substantia nigra of &agr;‐SYO‐injected mice compared to control. In conclusion, i.c.v. infusion of &agr;‐SYOs recapitulated some of PD‐associated non‐motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive‐like behavior typical of the disease. Moreover, &agr;‐SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to &agr;‐syn oligomers as the proximal neurotoxins responsible for early non‐motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.

The diabetes drug liraglutide reverses cognitive impairment in mice and attenuates insulin receptor and synaptic pathology in a non-human primate model of Alzheimer's disease: Liraglutide protects memory, insulin receptors, and synapses

Alzheimers disease (AD) is a devastating neurological disorder that still lacks an effective treatment, and this has stimulated an intense pursuit of disease‐modifying therapeutics. Given the increasingly recognized link between AD and defective brain insulin signaling, we investigated the actions of liraglutide, a glucagon‐like peptide‐1 (GLP‐1) analog marketed for treatment of type 2 diabetes, in experimental models of AD. Insulin receptor pathology is an important feature of AD brains that impairs the neuroprotective actions of central insulin signaling. Here, we show that liraglutide prevented the loss of brain insulin receptors and synapses, and reversed memory impairment induced by AD‐linked amyloid‐β oligomers (AβOs) in mice. Using hippocampal neuronal cultures, we determined that the mechanism of neuroprotection by liraglutide involves activation of the PKA signaling pathway. Infusion of AβOs into the lateral cerebral ventricle of non‐human primates (NHPs) led to marked loss of insulin receptors and synapses in brain regions related to memory. Systemic treatment of NHPs with liraglutide provided partial protection, decreasing AD‐related insulin receptor, synaptic, and tau pathology in specific brain regions. Synapse damage and elimination are amongst the earliest known pathological changes and the best correlates of memory impairment in AD. The results illuminate mechanisms of neuroprotection by liraglutide, and indicate that GLP‐1 receptor activation may be harnessed to protect brain insulin receptors and synapses in AD.

Functional test of PCDHB11, the most human-specific neuronal surface protein.

BackgroundBrain-expressed proteins that have undergone functional change during human evolution may contribute to human cognitive capacities, and may also leave us vulnerable to specifically human diseases, such as schizophrenia, autism or Alzheimer’s disease. In order to search systematically for those proteins that have changed the most during human evolution and that might contribute to brain function and pathology, all proteins with orthologs in chimpanzee, orangutan and rhesus macaque and annotated as being expressed on the surface of cells in the human central nervous system were ordered by the number of human-specific amino acid differences that are fixed in modern populations.ResultsPCDHB11, a beta-protocadherin homologous to murine cell adhesion proteins, stood out with 12 substitutions and maintained its lead after normalizing for protein size and applying weights for amino acid exchange probabilities. Human PCDHB11 was found to cause homophilic cell adhesion, but at lower levels than shown for other clustered protocadherins. Homophilic adhesion caused by a PCDHB11 with reversion of human-specific changes was as low as for modern human PCDHB11; while neither human nor reverted PCDHB11 adhered to controls, they did adhere to each other. A loss of function in PCDHB11 is unlikely because intra-human variability did not increase relative to the other human beta-protocadherins.ConclusionsThe brain-expressed protein with the highest number of human-specific substitutions is PCDHB11. In spite of its fast evolution and low intra-human variability, cell-based tests on the only proposed function for PCDHB11 did not indicate a functional change.