Ines Moreno-Gonzalez

Inflammatory Response in the Hippocampus of PS1M146L/APP751SL Mouse Model of Alzheimer's Disease: Age-Dependent Switch in the Microglial Phenotype from Alternative to Classic

Although the microglial activation is concomitant to the Alzheimers disease, its precise role (neuroprotection vs neurodegeneration) has not yet been resolved. Here, we show the existence of an age-dependent phenotypic change of microglial activation in the hippocampus of PS1xAPP model, from an alternative activation state with Aβ phagocytic capabilities (at 6 months) to a classic cytotoxic phenotype (expressing TNF-α and related factors) at 18 months of age. This switch was coincident with high levels of soluble Aβ oligomers and a significant pyramidal neurodegeneration. In vitro assays, using astromicroglial cultures, demonstrated that oligomeric Aβ42 and soluble extracts from 18-month-old PS1xAPP hippocampus produced a potent TNF-α induction whereas monomeric Aβ42 and soluble extract from 6- or 18-month-old control and 6-month-old PS1xAPP hippocampi produced no stimulation. This stimulatory effect was avoided by immunodepletion using 6E10 or A11. In conclusion, our results show evidence of a switch in the activated microglia phenotype from alternative, at the beginning of Aβ pathology, to a classical at advanced stage of the disease in this model. This change was induced, at least in part, by the age-dependent accumulation of extracellular soluble Aβ oligomers. Finally, these cytotoxic activated microglial cells could participate in the neuronal lost observed in AD.

Early neuropathology of somatostatin/NPY GABAergic cells in the hippocampus of a PS1 × APP transgenic model of Alzheimer's disease

At advanced stages, Alzheimers disease (AD) is characterized by an extensive neuronal loss. However, the early neurodegenerative deficiencies have not been yet identified. Here we report an extensive, selective and early neurodegeneration of the dendritic inhibitory interneurons (oriens-lacunosum moleculare, O-LM, and hilar perforant path-associated, HIPP, cells) in the hippocampus of a transgenic PS1xAPP AD model. At 6 months of age, from 22 different pre- and postsynaptic mRNA markers tested (including GABAergic, glutamatergic and cholinergic markers), only the expression of somatostatin (SOM) and NPY neuropeptides (O-LM and HIPP markers) displayed a significant decrease. Stereological cell counting demonstrated a profound diminution (50-60%) of SOM-immunopositive neurons, preceding the pyramidal cell loss in this AD model. SOM population co-expressing NPY was the most damaged cell subset. Furthermore, a linear correlation between SOM and/or NPY deficiency and Abeta content was also observed. Though the molecular mechanism of SOM neuronal loss remains to be determined, these findings might represent an early hippocampal neuropathology. Therefore, SOM and NPY neuropeptides could constitute important biomarkers to assess the efficacy of potential early AD treatments.

Misfolded protein aggregates: Mechanisms, structures and potential for disease transmission

Some of the most prevalent human degenerative diseases appear as a result of the misfolding and aggregation of proteins. Compelling evidence suggest that misfolded protein aggregates play an important role in cell dysfunction and tissue damage, leading to the disease. Prion protein (Prion diseases), amyloid-beta (Alzheimers disease), alpha-synuclein (Parkinsons disease), Huntingtin (Huntingtons disease), serum amyloid A (AA amyloidosis) and islet amyloid polypeptide (type 2 diabetes) are some of the proteins that trigger disease when they get misfolded. The recent understanding of the crucial role of misfolded proteins as well as the structural requirements and mechanism of protein misfolding have raised the possibility that these diseases may be transmissible by self-propagation of the protein misfolding process in a similar way as the infamous prions transmit prion diseases. Future research in this field should aim to clarify this possibility and translate the knowledge of the basic disease mechanisms into development of novel strategies for early diagnosis and efficient treatment.

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1M146L/APP751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.

Cross-seeding of misfolded proteins: implications for etiology and pathogenesis of protein misfolding diseases.

Accumulation of misfolded protein aggregates is a hallmark event in diverse diseases. These structures are able to seed their own polymerization by acting as aggregation nuclei both in vitro and in vivo. Recent studies in animal models suggest that misfolded proteins associated with different diseases can synergize in a heterologous fashion, potentiating pathological mechanisms and accelerating disease progression. The coexistence of misfolded protein aggregates has been described in patients affected by several protein misfolding disorders, suggesting a possible molecular cross-talk between pathological processes associated with different diseases. One putative mechanism for this cross-talk is a direct interaction between misfolded proteins, leading to cross-seeding of protein aggregation. This article summarizes the evidence for the cross-seeding phenomenon recently obtained in studies performed in vitro, in animal models, and in human patients, as well as the potential contribution of this mechanism to our understanding of the still elusive etiology and progression of maladies such as Alzheimers disease, where no effective diagnostic or therapeutic strategies exist.

Calretinin Interneurons are Early Targets of Extracellular Amyloid-β Pathology in PS1/AβPP Alzheimer Mice Hippocampus

Specific neuronal networks are preferentially affected in the early stages of Alzheimers disease (AD). The distinct subpopulations of hippocampal inhibitory GABAergic system have been shown to display differential vulnerability to neurodegeneration in AD. We have previously reported a substantial loss of SOM/NPY interneurons, whereas those expressing parvalbumin were unaltered, in the hippocampus of 6 month-old PS1/AbetaPP transgenic mice. In the present study, we now investigated the pathological changes of hippocampal calretinin (CR) interneurons in this PS1/AbetaPP model from 2 to 12 months of age. The total number of CR-immunoreactive inhibitory cells was determined by stereology in CA1 and CA2/3 subfields. Our findings show a substantial decrease (35%-45%) of CR-positive interneurons in both hippocampal subfields of PS1/AbetaPP mice at very early age (4 months) compared to age-matched control mice. This decrease was accompanied by a reduced CR mRNA content as determined by quantitative RT-PCR. However, the number of another hippocampal CR-positive population (belonging to Cajal-Retzius cells) was not affected. The selective early loss of CR-interneurons was parallel to the appearance of extracellular Abeta deposits, preferentially in CR-axonal fields, and the formation of dystrophic neurites. This specific GABAergic subpopulation plays a crucial role in the generation of synchronous rhythmic activity in hippocampus by controlling other interneurons. Therefore, early alterations of hippocampal inhibitory functionality in AD, caused by select CR-cells neurodegeneration, could result in cognitive impairments seen in initial stages of the disease.

Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons.

Increased neuroinflammatory reaction is frequently observed during normal brain aging. However, a direct link between neuroinflammation and neurodegeneration during aging has not yet been clearly shown. Here, we have characterized the age‐related hippocampal inflammatory processes and the potential relation with hippocampal neurodegeneration. The mRNA expression of the pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐α (TNF‐α), and the iNOs enzyme was significantly increased in aged hippocampus. Accordingly, numerous activated microglial cells were observed in aged rats. These cells were differentially distributed along the hippocampus, being more frequently located in the hilus and the CA3 area. The mRNA expression of somatostatin, a neuropeptide expressed by some GABAergic interneurons, and the number of somatostatin‐immunopositive cells decreased in aged rats. However, the number of hippocampal parvalbumin‐containing GABAergic interneurons was preserved. Interestingly, in aged rats, the mRNA expression of somatostatin and IL‐1β was inversely correlated and, the decrease in the number of somatostatin‐immunopositive cells was higher in the hilus of dentate gyrus than in the CA1 region. Finally, intraperitoneal chronic lipopolysaccharide (LPS) injection in young animals mimicked the age‐related hippocampal inflammation as well as the decrease of somatostatin mRNA expression. Present results strongly support the neuroinflammation as a potential factor involved in the age‐related degeneration of somatostatin GABAergic cells.

Smoking exacerbates amyloid pathology in a mouse model of Alzheimer’s disease

Several epidemiological studies have shown that cigarette smoking might alter the incidence of Alzheimers disease. However, inconsistent results have been reported regarding the risk of Alzheimers disease among smokers. Previous studies in experimental animal models have reported that administration of some cigarette components (for example, nicotine) alters amyloid-β aggregation, providing a possible link. However, extrapolation of these findings towards the in vivo scenario is not straightforward as smoke inhalation involves a number of other components. Here, we analysed the effect of smoking under more relevant conditions. We exposed transgenic mouse models of Alzheimers disease to cigarette smoke and analysed the neuropathological alterations in comparison with animals not subjected to smoke inhalation. Our results showed that smoking increases the severity of some abnormalities typical of Alzheimers disease, including amyloidogenesis, neuroinflammation and tau phosphorylation. Our findings suggest that cigarette smoking may increase Alzheimers disease onset and exacerbate its features and thus, may constitute an important environmental risk factor for Alzheimers disease.

Extracellular Amyloid-β and Cytotoxic Glial Activation Induce Significant Entorhinal Neuron Loss in Young PS1M146L/APP751SL Mice

Here we demonstrated that extracellular, not intracellular, amyloid-beta (Abeta) and the associated cytotoxic glial neuroinflammatory response are major contributors to early neuronal loss in a PS1xAPP model. A significant loss of principal (27%) and SOM/NPY (56-46%) neurons was found in the entorhinal cortex at 6 months of age. Loss of principal cells occurred selectively in deep layers (primarily layer V) whereas SOM/NPY cell loss was evenly distributed along the cortical column. Neither layer V pyramidal neurons nor SOM/NPY interneurons displayed intracellular Abeta immunoreactivity, even after formic acid retrieval; thus, extracellular factors should be preferentially implicated in this selective neurodegeneration. Amyloid deposits were mainly concentrated in deep layers at 4-6 months, and of relevance was the existence of a potentially cytotoxic inflammatory response (TNFalpha, TRAIL, and iNOS mRNAs were upregulated). Moreover, non-plaque associated activated microglial cells and reactive astrocytes expressed TNFalpha and iNOS, respectively. At this age, in the hippocampus of same animals, extracellular Abeta induced a non-cytotoxic glial activation. The opposite glial activation, at the same chronological age, in entorhinal cortex and hippocampus strongly support different mechanisms of disease progression in these two regions highly affected by Abeta pathology.

Molecular interaction between type 2 diabetes and Alzheimer’s disease through cross-seeding of protein misfolding

Numerous epidemiological studies have shown a significantly higher risk for development of Alzheimer’s disease (AD) in patients affected by type 2 diabetes (T2D), but the molecular mechanism responsible for this association is presently unknown. Both diseases are considered protein misfolding disorders associated with the accumulation of protein aggregates; amyloid-beta (Aβ) and tau in the brain during AD, and islet amyloid polypeptide (IAPP) in pancreatic islets in T2D. Formation and accumulation of these proteins follows a seeding-nucleation model, where a misfolded aggregate or ‘seed’ promotes the rapid misfolding and aggregation of the native protein. Our underlying hypothesis is that misfolded IAPP produced in T2D potentiates AD pathology by cross-seeding Aβ, providing a molecular explanation for the link between these diseases. Here, we examined how misfolded IAPP affects Aβ aggregation and AD pathology in vitro and in vivo. We observed that addition of IAPP seeds accelerates Aβ aggregation in vitro in a seeding-like manner and the resulting fibrils are composed of both peptides. Transgenic animals expressing both human proteins exhibited exacerbated AD-like pathology compared with AD transgenic mice or AD transgenic animals with type 1 diabetes (T1D). Remarkably, IAPP colocalized with amyloid plaques in brain parenchymal deposits, suggesting that these peptides may directly interact and aggravate the disease. Furthermore, inoculation of pancreatic IAPP aggregates into the brains of AD transgenic mice resulted in more severe AD pathology and significantly greater memory impairments than untreated animals. These data provide a proof-of-concept for a new disease mechanism involving the interaction of misfolded proteins through cross-seeding events which may contribute to accelerate or exacerbate disease pathogenesis. Our findings could shed light on understanding the linkage between T2D and AD, two of the most prevalent protein misfolding disorders.