Conference Agenda

Alzheimer’s disease (AD), affecting nearly 55 million people worldwide, represents an urgent yet unmet challenge for modern society, calling for exploring innovative targets. Astrocytes are the primary homeostatic cells in the central nervous system and represent a promising cell target. Indeed, at the early AD stage, reactive gliosis and neuroinflammation are associated with calcineurin (CaN) activation in astrocytes. CaN is a serine-threonine phosphatase responsible for Ca²⁺ signal-decoding in astrocytes, and its dysregulation appears to be a disease crucial event. Some evidence suggests that CaN modulation may interfere with AD pathology. Therefore, we investigated if deletion of the regulatory CaNB1 subunit of CaN in astrocytes could mitigate AD-related memory deficits, neuropathology, and neuroinflammation. We have generated two, acute and chronic, AD mouse models with astrocytic CaNB1 ablation (ACN-KO). We evaluated the ability of β-amyloid oligomers (AβOs) to impair memory and activate glial cells once injected into the cerebral ventricle of conditional ACN-KO mice. Next, we generated a tamoxifen-inducible astrocyte-specific CaNB1 knock-out in 3xTg-AD mice (indACNKO-AD). CaNB1 was deleted, by tamoxifen injection, in 11.7-month-old 3xTg-AD mice for 4.7 months. Spatial memory was evaluated using the Barnes maze; β-amyloid plaques burden, neurofibrillary tangle deposition, reactive gliosis, and neuroinflammation were assessed. The acute model showed that ICV injected AβOs in 2-month-old wild-type mice impaired recognition memory and fostered a pro-inflammatory microglia phenotype, whereas, in ACN-KO mice, AβOs were inactive. In indACNKO-AD mice, 4.7 months after CaNB1 depletion we found the preservation of spatial memory and cognitive flexibility, abolishment of amyloidosis, and reduction of neurofibrillary tangles, gliosis, and neuroinflammation. Our results suggest that ACN is crucial for the development of cognitive impairment, AD neuropathology, and neuroinflammation. Astrocyte-specific CaNB1 deletion is beneficial for both the abolishment of AβO-mediated detrimental effects and the treatment of ongoing AD-related pathology, hence representing an intriguing target for AD therapy.

The blood brain-barrier is an important structure limiting the access of pathological solutes in the central nervous system, but can also be considered as a proper obstacle, for example reducing the access of therapeutical drugs. Recently, the involvement of the BBB in the pathogenesis of several neurodegenerative disorders has been proven. Inhibiting BBB permeability can decrease the access of a series of deleterious circulating factors, including immune cells, inflammatory cytokines, and ions. Endothelial cells are the main cellular component of the BBB, but they are supported by astrocytes and microglia that actively modulate barriers properties both in physiological and pathological conditions. The use of an in vitro BBB model can result in an easier evaluation of the interplay between the different cellular components of the BBB. Here, using BAF-312, a sphingosine-1 phosphate (S1P) receptor modulator, already approved for the treatment of multiple sclerosis, we tried to better dissect the interaction occurring between endothelial cells, astrocytes and microglia, using human derived cell lines. The exposure to BAF-312 was able to directly affect endothelial cells properties, reducing the inflammatory-induced loss of tight junctions and preventing barrier permeability. Accordingly, BAF-312 indirectly affected endothelial properties, modulating astrocytic expression of inflammatory cytokines and microglial migration towards the endothelial/astrocytes co-cultures. These data suggest that the BBB and the crosstalk between its cellular components may represent an interesting pharmacological target in neurological disorders.

Growing evidence suggests a prominent role of the immune system in ALS pathoprogression, the most common and fatal adult-onset neuromuscular disorder.

We previously reported that MCP1/CCL2, one of the most potent pro-inflammatory chemokines, is strongly upregulated in the nervous system of C57 slow-progressing than 129Sv fast-progressing SOD1^G93A mice, the latter showing a poor immune response and eventual massive peripheral degeneration.

Given the pivotal role of MCP1-mediated signalling in driving damaged axons and muscle regeneration, we boosted the chemokine along the motor unit of the two SOD1^G93A models through a single intramuscular injection of a scAAV9 vector engineered with the Mcp1 gene.

Our observations revealed that the treatment had the opposite effect on the clinical phenotype of the two ALS models. C57SOD1^G93A mice responded positively to MCP1 boosting, anticipating the recruitment and phenotypic switch of leucocytes within the peripheral compartment. This sustained the activation of the myogenic programme and nerve regeneration, finally slackening off the motor symptoms progression. Conversely, 129SvSOD1^G93A mice exhibited a delayed and exacerbated activation of the pro-inflammatory immune muscle response upon MCP1 boosting, resulting in worsened motor ability late in the disease.

We provided direct evidence underlying the pivotal role of the immune response in driving skeletal muscle regeneration, spotlighting its nature and temporal activation as limiting factors to preserve the periphery and interfere with the ALS course tangibly.

Intriguingly, our data showed a novel immune-unrelated role of MCP1 in promoting motor axon regeneration and modulating neuroinflammation in the nervous system of SOD1^G93A mice, with the overall effect of reducing neurodegeneration.

Altogether, these observations highlight the immune response as a critical determinant for disease variability and proffer a reasonable explanation for the failure of systemic immunomodulatory treatments suggesting new potential strategies to hamper ALS progression.

The bidirectional communication between the central nervous system and gut bacteria has been demonstrated to play a key role in the pathogenesis of several brain pathologies, including Alzheimer’s Disease. Moreover, it has been suggested that probiotics may have potential beneficial effects on the immune system by changing microbiome composition and ultimately on cognition.

Here we assessed the role of GMB and of inflammation in the pathogenesis of Alzheimer’s Disease and in the potential benefits of probiotics.

Thirty-seven AD patients and sixty-two age-matched subjects with normal cognitive performance were recruited and stool and blood samples were collected. Moreover, the group of AD patients received a probiotic administration for 3 months and stool and blood samples were also collected at the end of the treatment. Pro-inflammatory cytokines and CRP were measured in plasma samples. Bacterial composition of fecal samples was analysed by sequencing the16S gene and the levels of the main short chain fatty acids (SCFAs) were measured by mass spectrometry while adhesion molecules by Fluorescence Activated Cell Sorting.

Higher levels of IL-6, IFN- ϒ, TNFα, IL-1b, IL-12p70 and IL-2 along with lower levels of the anti-inflammatory cytokine IL-10 were found in AD patients as compared to controls (all p<0.05). Three months treatment with probiotic supplement induced a significant reduction of IL-6, IFN-gamma levels and an increase in the levels of IL-10 (all p<0.05). Moreover, we found that changes in IFN-gamma and in IL-10 correlated significantly with changes in Ruminococcus, Rikenella, Escherichia-Shigella abundances and of Ruminococcaceae, Lachnospiraceae taxa (all p<0.05).

Our data overall indicate that AD patients show a higher inflammatory status compared to controls and that administration of a probiotics is pushing down the inflammatory status, via changes in microbiome composition and in microbial products, suggesting that probiotics could be beneficial in delaying neurodegeneration and cognitive deficits in subjects at risk of developing AD.