A Randomized, Double-Blind, and Sham-Controlled Trial of an Innovative Brain-Gut Photobiomodulation Therapy: Safety and Patient Compliance.

BACKGROUND: Recent innovative non-pharmacological interventions and neurostimulation devices have shown potential for application in the treatment of Alzheimer's disease (AD). These include photobiomodulation (PBM) therapy. OBJECTIVE: This pilot study assesses the safety, compliance with, and efficacy of a brain-gut PBM therapy for mild-to-moderate AD patients. METHODS: This double-blind, randomized, monocentric sham-controlled study started in 2018 and ended prematurely in 2020 due to the COVID-19 pandemic. Fifty-three mild-to-moderate AD patients were randomized, 27 in the PBM group and 26 in the sham group. All patients had 40 treatment sessions lasting 25 min each over 8 weeks and were followed for 4 weeks afterwards. Compliance with the treatment was recorded. Safety was assessed by recording adverse events (AEs), and efficacy was evaluated using neuropsychological tests. RESULTS: The PBM therapy proved to be safe in regard to the number of recorded AEs (44% of the patients), which were balanced between the PBM and sham groups. AEs were mainly mild, and no serious AEs were reported. The majority of the patients (92.5%) were highly compliant, which confirms the feasibility of the PBM treatment. Compared to the sham patients, the PBM patients showed lower ADAS-Cog comprehension subscores, higher forward verbal spans, and lower TMT-B execution times, which suggests an improvement in cognitive functions. CONCLUSION: This study demonstrates the tolerability of and patient compliance with a PBM-based treatment for mild-to-moderate AD patients. It highlights encouraging efficacy trends and provides insights for the design of the next phase trial in a larger AD patient sample.

Biomass measurements of single neurites in vitro using optical wavefront microscopy.

Quantitative phase microscopies (QPMs) enable label-free, non-invasive observation of living cells in culture, for arbitrarily long periods of time. One of the main benefits of QPMs compared with fluorescence microscopy is the possibility to measure the dry mass of individual cells or organelles. While QPM dry mass measurements on neural cells have been reported this last decade, dry mass measurements on their neurites has been very little addressed. Because neurites are tenuous objects, they are difficult to precisely characterize and segment using most QPMs. In this article, we use cross-grating wavefront microscopy (CGM), a high-resolution wavefront imaging technique, to measure the dry mass of individual neurites of primary neurons in vitro. CGM is based on the simple association of a cross-grating positioned in front of a camera, and can detect wavefront distortions smaller than a hydrogen atom (∼0.1 nm). In this article, an algorithm for dry-mass measurement of neurites from CGM images is detailed and provided. With objects as small as neurites, we highlight the importance of dealing with the diffraction rings for proper image segmentation and accurate biomass measurements. The high precision of the measurements we obtain using CGM and this semi-manual algorithm enabled us to detect periodic oscillations of neurites never observed before, demonstrating the sufficient degree of accuracy of CGM to capture the cell dynamics at the single neurite level, with a typical precision of 2%, i.e., 0.08 pg in most cases, down to a few fg for the smallest objects.

[Dental stem cells: myth or hope in neuroregenerative medicine?] / Cellules souches dentaires : mythe ou espoir en médecine neurorégénératrice ?

The use of dental stem cells has raised many hopes in the development of new treatments for neurodegenerative diseases. According to current statistics, about 1 in 6 people in the world would be affected by a neurological disease. This number continues to increase as the world's population ages, making neurodegenerative diseases probably the one of the major challenges of public health in the 21st century. Neurodegenerative diseases are characterized mainly by a progressive loss of cognitive abilities and patient autonomy related to loss and degeneration of neurons in brain structures. Unfortunately, today, the only treatments available for this type of disease do only relieve the symptoms, they do not treat them, and few clinical trials have been truly convincing to date. Hence, hope lies for these diseases in the development of other therapeutic approaches. As such, dental stem cells could be a promising area of research because of their rapid growth, their great capacity for differentiation into different types of cells (among neuronal ones for some of them) and how easy they can be obtained, without raising ethical issues as for example for embryonic stem cells.

Dental stem cells as a promising source for cell therapies in neurological diseases.

One of the main hallmarks of neurodegenerative diseases is the loss of neurons in the brain and/or spinal cord. The clinical characteristics of neurodegenerative diseases depend on the specific regions of the central nervous system that have undergone cell loss. These diseases place an enormous burden on society due to the degree of human suffering and their substantial economic cost. Indeed, approximately 1 in 6 individuals worldwide suffer from neurological disorders. As the world's population ages, the impact of neurological disorders is expected to increase and likely become one of the main public health and medical challenges of the coming century. There is still no cure for neurodegenerative diseases and currently available therapies only provide symptomatic relief. Hence, there is a pressing need to identify alternative therapeutic approaches to treat neurodegenerative diseases. Considering the global impact of these diseases and the need for new therapies, stem cell therapies have emerged as a promising treatment for neurodegenerative diseases. Notably, dental stem cells are an optimal candidate for cell-based therapeutic approaches, due primarily to the ease with which they can be obtained and their high regenerative potential. In this review, we summarize the potential of dental stem cells for use as a neurodegenerative disease therapy.

Cell-based therapy against prion diseases.

Despite multiple efforts to find treatments, prion diseases are still incurable. The currently available therapeutic strategies are mostly based on compounds to inhibit pathological PrP (PrPSc) accumulation, and cellular PrP (PrPC) conversion into PrPSc. However, they cannot reverse the pathological changes already present in the brain. Cell-based therapeutic strategies could promote the repair of the pre-existing brain damage. The few available data come mostly from preclinical studies using neural stem cells, bone marrow-derived microglia and mesenchymal stem cells, as cell sources. Moreover, the benefits of cell-based therapeutic strategies could be linked not only to the replacement of damaged cells, but also to the secretion of trophic factors by the grafted cells that might modulate inflammation, cell death, or endogenous neurogenesis.

Deuterium-reinforced linoleic acid lowers lipid peroxidation and mitigates cognitive impairment in the Q140 knock in mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease which has no effective treatment and is characterized by psychiatric disorders, motor alterations, and dementia, with the cognitive deficits representing a devastating aspect of the disorder. Oxidative stress and elevated levels of lipid peroxidation (LPO) products are found in mouse models and patients with HD, suggesting that strategies to reduce LPO may be beneficial in HD. In contrast with traditional antioxidants, substituting hydrogen with deuterium at bis-allylic sites in polyunsaturated fatty acids (D-PUFA) decreases the rate-limiting initiation step of PUFA autoxidation, a strategy that has shown benefits in other neurodegenerative diseases. Here, we investigated the effect of D-PUFA treatment in a knock-in mouse model of HD (Q140) which presents motor deficits and neuropathology from a few months of age, and progressive cognitive decline. Q140 knock-in mice were fed a diet containing either D- or H-PUFAs for 5 months starting at 1 month of age. D-PUFA treatment significantly decreased F2 -isoprostanes in the striatum by approximately 80% as compared to H-PUFA treatment and improved performance in novel object recognition tests, without significantly changing motor deficits or huntingtin aggregation. Therefore, D-PUFA administration represents a promising new strategy to broadly reduce rates of LPO, and may be useful in improving a subset of the core deficits in HD.

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.

Assessing a multiplex-targeted proteomics approach for the clinical diagnosis of periodontitis using saliva samples.

AIM: The present study focused on the research of new biomarkers based on the liquid chromatography-multiple-reaction monitoring (MRM) proteomic profile in whole saliva of patients with periodontitis compared with periodontal healthy patients. METHODS: A 30-min multiplexed liquid chromatography-MRM method was used for absolute quantification of 35 plasma biomarkers in saliva from control patients and patients with periodontitis. RESULTS: Three proteins namely hemopexin, plasminogen and α-fibrinogen were shown to be clearly related to the presence of periodontitis compared with healthy patients. Apolipoprotein H was found to discriminate for the first time chronic and aggressive periodontitis. CONCLUSION: Our results indicate that this innovative MRM method could be used to screen for periodontitis in clinical environment. Furthermore, apolipoprotein H was found to be a discriminant biomarker of aggressive periodontitis.

Prion diseases and adult neurogenesis: how do prions counteract the brain's endogenous repair machinery?

Scientific advances in stem cell biology and adult neurogenesis have raised the hope that neurodegenerative disorders could benefit from stem cell-based therapy. Adult neurogenesis might be part of the physiological regenerative process, however it might become impaired by the disease's mechanism and therefore contribute to neurodegeneration. In prion disorders this endogenous repair system has rarely been studied. Whether adult neurogenesis plays a role or not in brain repair or in the propagation of prion pathology remains unclear. We have recently investigated the status of adult neural stem cells isolated from prion-infected mice. We were able to show that neural stem cells accumulate and replicate prions thus resulting in an alteration of their neuronal destiny. We also reproduced these results in adult neural stem cells, which were infected in vitro. The fact that endogenous adult neurogenesis could be altered by the accumulation of misfolded prion protein represents another great challenge. Inhibiting prion propagation in these cells would thus help the endogenous neurogenesis to compensate for the injured neuronal system. Moreover, understanding the endogenous modulation of the neurogenesis system would help develop effective neural stem cell-based therapies.

Elements modulating the prion species barrier and its passage consequences.

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor. As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.

Systemic delivery of siRNA down regulates brain prion protein and ameliorates neuropathology in prion disorder.

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrP(C) level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.

Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: involvement of endogenous neural stem cells in prion diseases.

Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.

Stem cell therapy extends incubation and survival time in prion-infected mice in a time window-dependant manner.

Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.

Prion infection of differentiated neurospheres.

Until now only a few cell lines have been proved able to propagate prions and only limited prion strains have been replicated in cell models. Neurosphere lines isolated from the brains of mice at embryonic day 14 grow as aggregates and contain CNS stem cells. Others authors have previously reported that cultured neurospheres expressing cellular prion protein (PrP(C)) can be infected with prions. As potential neural progenitors the neurosphere cultures are supposed to differentiate into neurons and astrocytes which represent the main cell types infected by prions in vivo. Here we study the ability of undifferentiated and differentiated neurospheres to replicate several prion strains. Neurosphere cultures were isolated from 129/ola, FVB, Prnp(0/0) and Tga20 mice, which over-express murine PrP. We were not able to detect PrP(res) accumulation in dividing neurosphere cultures after prion exposure to two different mouse adapted scrapie inocula (RML and 22L). In contrast, with differentiated neurosphere cultures expressing PrP(C) (129/ola, FVB and Tga20) a successful PrP(Res) amplification was observed in very short time experiments when infected with the same inocula, implying that cell differentiation improve prion replication in these cultured cells. The mouse BSE adapted inocula (301C) was not amplified in these neurosphere cultures neither before nor after differentiation, suggesting that these cell cultures showed a differential prion strain susceptibility. These results suggest that differentiated neurosphere cultures can complement prion bioassays in mouse models.

The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals.

BACKGROUND: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity. RESULTS: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells. CONCLUSION: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues.