David Baglietto-Vargas

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.

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.

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.

Activity-Dependent Neuroprotective Protein (ADNP) Expression in the Amyloid Precursor Protein/Presenilin 1 Mouse Model of Alzheimer's Disease

A major determinant in the pathogenesis of Alzheimer’s disease (AD) is the deposition of β-amyloid (Aβ) peptides in specific areas of the central nervous system. Therefore, animal models of Alzheimer amyloidosis are excellent tools to identify candidates to facilitate drug screening and to understand the molecular pathology of AD. Activity-dependent neuroprotective protein (ADNP) plays an essential role in brain development, and NAP (NAPVSIPQ, generic name: davunetide)—a peptide derived from ADNP—is currently in clinical development for the treatment of neurodegenerative disorders. However, the link between ADNP expression and AD remains unexplored. To test whether ADNP is affected by the onset of AD and progression, we employed the PS1xAPP mouse model (PS1M146Lu2009×u2009APP751SL transgenic mice) to analyze the mRNA expression of ADNP in the hippocampus and cerebellum in early and advanced stages of disease. Results showed that ADNP expression in 6-month-old PS1xAPP mice hippocampus was higher than in wild-type (WT) mice. ADNP was originally identified as a vasoactive intestinal peptide (VIP)-responsive gene taking part in the VIP-mediated neurotrophic pathway. Interestingly, the expression of VIP was not affected in the same experimental setting, suggesting that ADNP expression is a VIP-independent marker associated with AD. Moreover, in the cerebellum, a brain area not affected by Aβ deposition, ADNP mRNA expression in 6-month-old PS1xAPP and WT were not different. A similar extent of hippocampal ADNP expression was observed in 18-month-old WT and PS1xAPP mice, in contrast to the differential expression level at 6xa0months of age. However, hippocampal ADNP expression in both WT and PS1xAPP was increased with aging similar to VIP mRNA expression. Our findings support the hypothesis that ADNP expression is related to early or mild AD progression by a VIP-independent mechanism.

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.

Segregation of two glutaminase isoforms in islets of Langerhans

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in beta-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in alpha-cells, LGA was very abundant in beta-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.

In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer's disease pathology.

BackgroundAlzheimer’s disease (AD) is characterized by the abnormal accumulation of extracellular beta-amyloid (Abeta) plaques, intracellular hyperphosphorylated tau, progressive synaptic alterations, axonal dystrophies, neuronal loss and the deterioration of cognitive capabilities of patients. However, no effective disease-modifying treatment has been yet developed. In this work we have evaluated whether chronic lithium treatment could ameliorate the neuropathology evolution of our well characterized PS1M146LxAPPSwe-London mice model.ResultsThough beneficial effects of lithium have been previously described in different AD models, here we report a novel in vivo action of this compound that efficiently ameliorated AD-like pathology progression and rescued memory impairments by reducing the toxicity of Abeta plaques. Transgenic PS1M146LxAPPSwe-London mice, treated before the pathology onset, developed smaller plaques characterized by higher Abeta compaction, reduced oligomeric-positive halo and therefore with attenuated capacity to induce neuronal damage. Importantly, neuronal loss in hippocampus and entorhinal cortex was fully prevented. Our data also demonstrated that the axonal dystrophic area associated with lithium-modified plaques was highly reduced. Moreover, a significant lower accumulation of phospho-tau, LC3-II and ubiquitinated proteins was detected in treated mice. Our study highlights that this switch of plaque quality by lithium could be mediated by astrocyte activation and the release of heat shock proteins, which concentrate in the core of the plaques.ConclusionsOur data demonstrate that the pharmacological in vivo modulation of the extracellular Abeta plaque compaction/toxicity is indeed possible and, in addition, might constitute a novel promising and innovative approach to develop a disease-modifying therapeutic intervention against AD.

Inter-individual variability in the expression of the mutated form of hPS1M146L determined the production of Aβ peptides in the PS1xAPP transgenic mice

The detection of the early phenotypic modifications of Alzheimers disease (AD) models is fundamental to understand the progression and identify pharmacologic targets of this pathology. However, a large variability within different models and between age‐matched mice from the same model has been observed. This variability could be due to heterogeneity in the Aβ production. Present results showed the existence of a large variability in the Aβ deposition in both hippocampus and cortex in 6‐month‐old PS1xAPP mice. This variability was not due to the expression of hAPP751SL, however, linear relationship between hPS1M146L mRNA and Aβ production was identified. The Aβ content was related to the incorporation of the hPS1M146L into functional γ‐secretase complexes, detected by the presence of the corresponding human or endogenous PS1‐CTFs. Animals expressing low amount of hPS1M146L mRNA, displayed low hPS1‐CTF incorporation and produced a low amount of Aβ peptides. Conversely, mice with relatively high hPS1 mRNA expression displayed high hPS1‐CTF and high Aβ deposition. Furthermore, the Aβ total and Aβ1‐42 content was increased dramatically by the expression of hPS1M146L (as compared with transgenic APPsl littermates). Therefore, variations in the expression of transgenic form of hPS1M146L in this model, or even between different models, influenced strongly the incorporation of the mutated PS1 into functional γ‐secretase complexes, the production of Aβ peptides and, in consequence, the detrimental effects of Aβ peptides. These data might implicate an “apparent gain‐of‐function” of the γ‐secretase complex by the expression of the mutated PS1M146L.