Homogentisic acid induces autophagy alterations leading to chondroptosis in human chondrocytes: Implications in Alkaptonuria.

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.

Longevity-Associated Variant of BPIFB4 Confers Neuroprotection in the STHdh Cell Model of Huntington Disease.

Huntington's disease (HD) is caused by the production of mutant Huntingtin (mHTT), characterized by long polyglutamine repeats with toxic effects. There are currently no clinically validated therapeutic agents that slow or halt HD progression, resulting in a significant clinical unmet need. The striatum-derived STHdh cell line, generated from mHTT knock-in mouse embryos (STHdhQ111/Q111), represents a useful model to study mechanisms behind pathogenesis of HD and to investigate potential new therapeutic targets. Indeed, these cells show susceptibility to nucleolar stress, activated DNA damage response and apoptotic signals, and elevated levels of H3K9me3 that all together concur in the progressive HD pathogenesis. We have previously shown that the adeno-associated viral vector-mediated delivery of the longevity-associated variant (LAV) of BPIFB4 prevents HD progression in a mouse model of HD. Here, we show that LAV-BPIFB4 stably infected in STHdhQ111/Q111 cells reduces (i) nucleolar stress and DNA damage through the improvement of DNA repair machinery, (ii) apoptosis, through the inhibition of the caspase 3 death signaling, and (iii) the levels of H3K9me3, by accelerating the histone clearance, via the ubiquitin-proteasome pathway. These findings pave the way to propose LAV-BPIFB4 as a promising target for innovative therapeutic strategies in HD.

Alkaptonuria: From Molecular Insights to a Dedicated Digital Platform.

Alkaptonuria (AKU) is a genetic disorder that affects connective tissues of several body compartments causing cartilage degeneration, tendon calcification, heart problems, and an invalidating, early-onset form of osteoarthritis. The molecular mechanisms underlying AKU involve homogentisic acid (HGA) accumulation in cells and tissues. HGA is highly reactive, able to modify several macromolecules, and activates different pathways, mostly involved in the onset and propagation of oxidative stress and inflammation, with consequences spreading from the microscopic to the macroscopic level leading to irreversible damage. Gaining a deeper understanding of AKU molecular mechanisms may provide novel possible therapeutical approaches to counteract disease progression. In this review, we first describe inflammation and oxidative stress in AKU and discuss similarities with other more common disorders. Then, we focus on HGA reactivity and AKU molecular mechanisms. We finally describe a multi-purpose digital platform, named ApreciseKUre, created to facilitate data collection, integration, and analysis of AKU-related data.

The longevity-associated BPIFB4 gene guarantees vascular homeostasis and immune protection through platelets.

Beyond their activity in hemostasis and thrombosis, recent advances attribute platelets a pro-youthful role capable to attenuate immune senescence and age-related neuroinflammation. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models has proved strategic to cope with frailty conditions, aging-related events, e.g., cardiovascular ones, and immune dysfunction mainly through a favorable conditioning of the immune system. However, whether platelets participate in LAV-BPIFB4 therapeutic action is currently unknown. Herein, we discovered that platelets were instrumental in boosting the favorable health outcomes of the systemic AAV-LAV-BPIFB4 gene transfer in vivo, as the α-CD42b platelet depletion completely abolished the vascular protective action of LAV-BPIFB4 and suppressed its pro-resolutive CD206 + anti-/CD86 + pro-inflammatory Ly6C + monocyte skewing to LPS stimulation. Of note, this is associated with a huge drop in the protective levels of BPIFB4 in the plasma of AAV-LAV-BPIFB4-injected C57BL/6 mice, indicating that plasma circulating platelets may be a reservoir of the BPIFB4 protein. Indeed, we noticed that BPIFB4 was released by human platelets, a process that is amplified in LAV-allele carrier donors. Accordingly, lentivirus-mediated overexpression of human LAV-BPIFB4 isoform, but not WT-BPIFB4 isoform was able in leading differentiated megakaryocytes to release more platelet-like-particles enriched for BPIFB4. In addition, in vitro, the M2 macrophage polarization increased when releasate from platelets, and even more from LAV pre-stimulated once, was added in monocyte cell culture. Our data suggest that platelet release of BPIFB4 and of yet-to-be-determined unidentified factors mediates the therapeutic efficacy of LAV-BPIFB4 treatment.

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

AKUImg: A database of cartilage images of Alkaptonuria patients.

ApreciseKUre is a multi-purpose digital platform facilitating data collection, integration and analysis for patients affected by Alkaptonuria (AKU), an ultra-rare autosomal recessive genetic disease. We present an ApreciseKUre plugin, called AKUImg, dedicated to the storage and analysis of AKU histopathological slides, in order to create a Precision Medicine Ecosystem (PME), where images can be shared among registered researchers and clinicians to extend the AKU knowledge network. AKUImg includes a new set of AKU images taken from cartilage tissues acquired by means of a microscopic technique. The repository, in accordance to ethical policies, is publicly available after a registration request, to give to scientists the opportunity to study, investigate and compare such precious resources. AKUImg is also integrated with a preliminary but accurate predictive system able to discriminate the presence/absence of AKU by comparing histopatological affected/control images. The algorithm is based on a standard image processing approach, namely histogram comparison, resulting to be particularly effective in performing image classification, and constitutes a useful guide for non-AKU researchers and clinicians.