Studying atherosclerotic calcification in vivo requires mouse models with genetic modifications. Previous studies showed that injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice promotes atherosclerosis. We aimed to study cardiovascular calcification induced by PCSK9 AAV in C57BL/6J mice.

Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1-/-) murine primary hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN's neurotrophic effects.

Disuse is a cardinal sign of various neurological diseases like stroke, cerebral palsy, and amyotrophic lateral sclerosis. Disuse leads to reduced mechanical loading of the skeleton, and a substantial and significant loss of bone mass quickly materializes. Several animal models have been proposed to investigate the pathogenesis of disuse-induced bone loss and to test new pharmaceutical targets to counteract it. As animal models may overcome several of the limitations in observational studies conducted in patients and allow for measurements not possible in humans, the primary objective of the present study is to provide a comprehensive overview of the available animal models of disuse-induced bone loss.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

Osteopenia and sarcopenia develops rapidly during disuse. The study investigated whether intermittent parathyroid hormone (1-34) (PTH) and growth hormone (GH) administered alone or in combination could prevent or mitigate disuse osteopenia and sarcopenia in rats. Disuse was achieved by injecting 4IU botulinum toxin A (BTX) into the right hindlimb musculature of 12-14-week-old female Wistar rats. Seventy-two rats were divided into six groups: 1. Baseline; 2. Ctrl; 3. BTX; 4. BTX+GH; 5. BTX+PTH; 6. BTX+PTH+GH. PTH (1-34) (60μg/kg/day) and GH (5mg/kg/day). The animals were sacrificed after 6weeks of treatment. Sarcopenia was established by histomorphometry, while the skeletal properties were determined using DXA, μCT, mechanical testing, and dynamic bone histomorphometry. Disuse resulted in lower muscle mass (-63%, p<0.05), trabecular BV/TV (-28%, p<0.05), Tb.Th (-11%, p<0.05), lower diaphyseal cortical thickness (-10%, p<0.001), and lower bone strength at the distal femoral metaphysis (-27%, p<0.001) compared to Ctrl animals. PTH fully counteracted the immobilization-induced lower BV/TV, Tb.Th, and distal femoral metaphyseal strength. GH increased muscle mass (+17%, p<0.05) compared to BTX, but did not prevent the immobilization-induced loss of bone strength, BV/TV, and cortical trabecular thickness. Combination of PTH and GH increased distal femoral metaphyseal bone strength (+45%, p<0.001), BV/TV (+50%, p<0.05), Tb.Th (+40%, p<0.05), and whole femoral aBMD (+15%, p<0.001) compared to BTX and muscle mass (+21%, p<0.05) compared to BTX+PTH. In conclusion, PTH and GH in combination is more efficient at preventing the disuse-related deterioration of bone strength, density, and micro-architecture than either PTH or GH given as monotherapy. Furthermore, GH, either alone or in combination with PTH, attenuated disuse-induced loss of muscle mass. The combination of PTH and GH resulted in a more effective treatment than PTH and GH as monotherapy.

Damage of the lower motor neuron cell bodies or their axons results in reduced or abolished voluntary movement accompanied by a substantial loss of bone and muscle mass. Intermittent parathyroid hormone 1-34 (PTH) (teriparatide) is one of the most potent bone-anabolic treatment regimens. ActRIIA-mFc is an activin type IIA decoy receptor that increases bone mass mediated by inhibition of the activin receptor signaling pathway. We investigated whether PTH or ActRIIA-mFc alone or in combination could prevent loss of bone and muscle mass induced by injecting botulinum toxin A (BTX) into the right hind limb in mice. Seventy-two 16-week-old female C57BL/6 mice were allocated to the following groups: Baseline, Control, BTX, BTX + ActRIIA-mFc (10 mg/kg), BTX + PTH (100 μg/kg), and BTX + ActRIIA-mFc + PTH. The mice were sacrificed after three weeks of disuse and treatment. In contrast to monotherapy with PTH, ActRIIA-mFc alone or in combination with PTH was able partly or completely to prevent disuse-induced loss of whole femoral bone mass, trabecular thickness, and bone strength. Moreover, an additive effect of ActRIIA-mFc and PTH on areal bone mineral density and trabecular bone volume was found. In summary, ActRIIA-mFc and PTH in combination were more effective in preventing disuse-induced bone loss and deterioration of trabecular micro-architecture than either treatment alone.

Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group: 1. Baseline; 2. Normobaric, 4 weeks; 3. Hypobaric hypoxia, 4 weeks; 4. Normobaric, 8 weeks; 5. Hypobaric hypoxia, 8 weeks; 6. Normobaric, 12 weeks; and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased. Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation+Zln, Recovery, Recovery+Zln, and Virgin Control (age-matched). The lactation period was 12days, then the pups were removed, and thereafter recovery took place for 28days. Zln, 100μg/kg, was given s.c. on the day of delivery, and again 4 and 8days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (-55% vs. Pregnant, p<0.01), BV/TV (-40% vs. Pregnant, p<0.01), and trabecular thickness (Tb.Th) (-29% vs. Pregnant, p<0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+45%, p<0.01) and Tb.Th (+16%, p<0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.

Immobilization causes rapid and massive bone loss. By comparing Botulinum Toxin A (BTX)-induced bone loss in mouse strains with different genetic backgrounds we investigated whether the genetic background had an influence on the severity of the osteopenia. Secondly, we investigated whether BTX had systemic effects on bone. Female mice from four inbred mouse strains (BALB/cJ, C57BL/6 J, DBA/2 J, and C3H/HeN) were injected unilaterally with BTX (n = 10/group) or unilaterally with saline (n = 10/group). Mice were euthanized after 21 days, and the bone properties evaluated using μCT, DXA, bone histomorphometry, and mechanical testing. BTX resulted in substantially lower trabecular bone volume fraction (BV/TV) and trabecular thickness in all mouse strains. The deterioration of BV/TV was significantly greater in C57BL/6 J (- 57%) and DBA/2 J (- 60%) than in BALB/cJ (- 45%) and C3H/HeN (- 34%) mice. The loss of femoral neck fracture strength was significantly greater in C57BL/6 J (- 47%) and DBA/2 J (- 45%) than in C3H (- 25%) mice and likewise the loss of mid-femoral fracture strength was greater in C57BL/6 J (- 17%), DBA/2 J (- 12%), and BALB/cJ (- 9%) than in C3H/HeN (- 1%) mice, which were unaffected. Using high resolution μCT we found no evidence of a systemic effect on any of the microstructural parameters of the contralateral limb. Likewise, there was no evidence of a systemic effect on the bone strength in any mouse strain. We did, however, find a small systemic effect on aBMD in DBA/2 J and C3H/HeN mice. The present study shows that BTX-induced immobilization causes the greatest loss of cortical and trabecular bone in C57BL/6 J and DBA/2 J mice. A smaller loss of bone microstructure and fracture strength was seen in BALB/cJ mice, while the bone microstructure and fracture strength of C3H/HeN mice were markedly less affected. This indicates that BTX-induced loss of bone is mouse strain dependent. We found only minimal systemic effects of BTX.

Individuals with type 1 diabetes mellitus are at high risk of developing atherosclerotic cardiovascular disease, but the underlying mechanisms by which type 1 diabetes accelerates atherosclerosis remain unknown. Increased retention of low-density lipoprotein (LDL) in atherosclerosis-prone sites of the diabetic vascular wall has been suggested, but direct evidence is lacking. In the present study, we investigated whether retention of LDL is increased in atherosclerotic-prone areas using a murine model of type 1 diabetes.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1beta is primarily neurotoxic in ischemic stroke, TNF-alpha may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

The trans-membrane protease serine 2 (TMPRSS2) is essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry and infection. Efficacy and safety of TMPRSS2 inhibitors in patients with coronavirus disease 2019 (Covid-19) have not been evaluated in randomized trials.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning.