Accelerated sarcopenia precedes learning and memory impairments in the P301S mouse model of tauopathies and Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) impairs cognitive functions and peripheral systems, including skeletal muscles. The PS19 mouse, expressing the human tau P301S mutation, shows cognitive and muscular pathologies, reflecting the central and peripheral atrophy seen in AD. METHODS: We analysed skeletal muscle morphology and neuromuscular junction (NMJ) through immunohistochemistry and advanced image quantification. A factorial Analysis of Variance assessed muscle weight, NCAM expression, NMJ, myofibre type distribution, cross-sectional areas, expression of single or multiple myosin heavy-chain isoforms, and myofibre grouping in PS19 and wild type (WT) mice over their lifespan (1-12 months). RESULTS: Significant weight differences in extensor digitorum longus (EDL) and soleus muscles between WT and PS19 mice were noted by 7-8 months. For EDL muscle in females, WT weighed 0.0113 ± 0.0005 compared with PS19's 0.0071 ± 0.0008 (P < 0.05), and in males, WT was 0.0137 ± 0.0001 versus PS19's 0.0069 ± 0.0006 (P < 0.005). Similarly, soleus muscle showed significant differences; females (WT: 0.0084 ± 0.0004; PS19: 0.0057 ± 0.0005, P < 0.005) and males (WT: 0.0088 ± 0.0003; PS19: 0.0047 ± 0.0004, P < 0.0001). Analysis of the NMJ in PS19 mice revealed a marked reduction in myofibre innervation at 5 months, with further decline by 10 months. NMJ pre-terminals in PS19 mice became shorter and simpler by 5 months, showing a steep decline by 10 months. Genotype and age strongly influenced muscle NCAM immunoreactivity, denoting denervation as early as 5-6 months in EDL muscle Type II fibres, with earlier effects in soleus muscle Type I and II fibres at 3-4 months. Muscle denervation and subsequent myofibre atrophy were linked to a reduction in Type IIB fibres in the EDL muscle and Type IIA fibres in the soleus muscle, accompanied by an increase in hybrid fibres. The EDL muscle showed Type IIB fibre atrophy with WT females at 1505 ± 110 µm2 versus PS19's 1208 ± 94 µm2, and WT males at 1731 ± 185 µm2 versus PS19's 1227 ± 116 µm2. Similarly, the soleus muscle demonstrated Type IIA fibre atrophy from 5 to 6 months, with WT females at 1194 ± 52 µm2 versus PS19's 858 ± 62 µm2, and WT males at 1257 ± 43 µm2 versus PS19's 1030 ± 55 µm2. Atrophy also affected Type IIX, I + IIA, and IIA + IIX fibres in both muscles. The timeline for both myofibre and overall muscle atrophy in PS19 mice was consistent, indicating a simultaneous decline. CONCLUSIONS: Progressive and accelerated neurogenic sarcopenia may precede and potentially predict cognitive deficits observed in AD.

Sex differences in single neuron function and proteomics profiles examined by patch-clamp and mass spectrometry in the locus coeruleus of the adult mouse.

AIMS: This study aimed to characterize the properties of locus coeruleus (LC) noradrenergic neurons in male and female mice. We also sought to investigate sex-specific differences in membrane properties, action potential generation, and protein expression profiles to understand the mechanisms underlying neuronal excitability variations. METHODS: Utilizing a genetic mouse model by crossing Dbhcre knock-in mice with tdTomato Ai14 transgenic mice, LC neurons were identified using fluorescence microscopy. Neuronal functional properties were assessed using patch-clamp recordings. Proteomic analyses of individual LC neuron soma was conducted using mass spectrometry to discern protein expression profiles. Data are available via ProteomeXchange with identifier PXD045844. RESULTS: Female LC noradrenergic neurons displayed greater membrane capacitance than those in male mice. Male LC neurons demonstrated greater spontaneous and evoked action potential generation compared to females. Male LC neurons exhibited a lower rheobase and achieved higher peak frequencies with similar current injections. Proteomic analysis revealed differences in protein expression profiles between sexes, with male mice displaying a notably larger unique protein set compared to females. Notably, pathways pertinent to protein synthesis, degradation, and recycling, such as EIF2 and glucocorticoid receptor signaling, showed reduced expression in females. CONCLUSIONS: Male LC noradrenergic neurons exhibit higher intrinsic excitability compared to those from females. The discernible sex-based differences in excitability could be ascribed to varying protein expression profiles, especially within pathways that regulate protein synthesis and degradation. This study lays the groundwork for future studies focusing on the interplay between proteomics and neuronal function examined in individual cells.

Brainstem noradrenergic neurons: Identifying a hub at the intersection of cognition, motility, and skeletal muscle regulation.

Brainstem noradrenergic neuron clusters form a node integrating efferents projecting to distinct areas such as those regulating cognition and skeletal muscle structure and function, and receive dissimilar afferents through established circuits to coordinate organismal responses to internal and environmental challenges. Genetic lineage tracing shows the remarkable heterogeneity of brainstem noradrenergic neurons, which may explain their varied functions. They project to the locus coeruleus, the primary source of noradrenaline in the brain, which supports learning and cognition. They also project to pre-ganglionic neurons, which lie within the spinal cord and form synapses onto post-ganglionic neurons. The synapse between descending brainstem noradrenergic neurons and pre-ganglionic spinal neurons, and these in turn with post-ganglionic noradrenergic neurons located at the paravertebral sympathetic ganglia, support an anatomical hierarchy that regulates skeletal muscle innervation, neuromuscular transmission, and muscle trophism. Whether any noradrenergic neuron subpopulation is more susceptible to damaged protein deposit and death with ageing and neurodegeneration is a relevant question that answer will help us to detect neurodegeneration at an early stage, establish prognosis, and anticipate disease progression. Loss of muscle mass and strength with ageing, termed sarcopenia, may predict impaired cognition with ageing and neurodegeneration and establish an early time to start interventions aimed at reducing central noradrenergic neurons hyperactivity. Complex multidisciplinary approaches, including genetic tracing, specific circuit labelling, optogenetics and chemogenetics, electrophysiology, and single-cell transcriptomics and proteomics, are required to test this hypothesis pre-clinical.

Skeletal muscle sympathetic denervation disrupts the neuromuscular junction postterminal organization: A single-cell quantitative approach.

The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.

Long-term, induced expression of Hand2 in peripheral sympathetic neurons ameliorates sarcopenia in geriatric mice.

BACKGROUND: The discovery of adrenoceptors, which mediate the effects of the sympathetic nervous system neurotransmitter norepinephrine on specific tissues, sparked the development of sympathomimetics that have profound influence on skeletal muscle mass. However, chronic administration has serious side effects that preclude their use for muscle-wasting conditions such as sarcopenia, the age-dependent decline in muscle mass, force, and power. Devising interventions that can adjust neurotransmitter release to changing physiological demands will require understanding how the sympathetic nervous system affects muscle motor innervation and muscle mass, which will prevent sarcopenia-associated impaired mobility, falls, institutionalization, co-morbidity, and premature death. Here, we tested the hypothesis that prolonged heart and neural crest derivative 2 (Hand2) expression in peripheral sympathetic neurons (SNs) ameliorates sympathetic muscle denervation, motor denervation, and sarcopenia in geriatric mice. METHODS: We delivered either a viral vector encoding the transcription factor Hand2 or an empty vector (EV) driven to SNs by the PRSx8 promoter by injecting the saphenous vein in 16-month-old C57BL/6 mice that were sacrificed 10-11 months later. Studies relied on sympathetic and muscle immunohistochemistry analysed by confocal microscopy, nerve and muscle protein expression assessed by immunoblots, nerve-evoked and muscle-evoked maximal muscle contraction force, extensor digitorum longus (EDL) muscle RNA sequencing, SN real-time PCR, and tests of physical performance using an inverted-cling grip test and in an open-arena setting. RESULTS: Examining the mice 10-11 months later, we found that inducing Hand2 expression in peripheral SNs preserved (i) the number of neurons (EV: 0.32 ± 0.03/µm2 , n = 6; Hand2: 0.92 ± 0.08/µm2 , n = 7; P < 0.0001) and size (EV: 279 ± 18 µm2 , n = 6; Hand2: 396 ± 18 µm2 , n = 7; P < 0.0001); (ii) lumbricalis muscle sympathetic innervation (EV: 1.4 ± 1.5 µm/µm2 , n = 5; Hand2: 12 ± 1.8 µm/µm2 , n = 5; P < 0.001); (iii) tibialis anterior, gastrocnemius, EDL, and soleus muscles weight and whole-body strength (EV: 48 ± 6.4 s, n = 6; Hand2: 102 ± 6.8 s, n = 6; P < 0.001); (iv) EDL type IIb, IIx, and II/IIx and soleus type I, IIa, IIx, IIa/IIx, and IIb/IIx myofibre cross-sectional area; (v) nerve-evoked (EV: 16 ± 2.7 mN; Hand2: 30 ± 4.4 mN; P < 0.001) and muscle-evoked (EV: 24 ± 3.8 mN, n = 5; Hand2: 38 ± 3.0 mN, n = 8; P < 0.001) muscle force by 150 Hz-3 s pulses; and (vi) motor innervation assessed by measuring presynaptic/postsynaptic neuromuscular junction area overlay. CONCLUSIONS: Preserving Hand2 expression in SNs from middle-aged to very old mice attenuates decreases in muscle mass and force by (i) maintaining skeletal muscle sympathetic and motor innervation, (ii) improving membrane and total acetylcholine receptor stability and nerve-evoked and muscle-evoked muscle contraction, (iii) preventing the elevation of inflammation and myofibrillar protein degradation markers, and (iv) increasing muscle autophagy.

The emerging role of the sympathetic nervous system in skeletal muscle motor innervation and sarcopenia.

Examining neural etiologic factors'role in the decline of neuromuscular function with aging is essential to our understanding of the mechanisms underlying sarcopenia, the age-dependent decline in muscle mass, force and power. Innervation of the skeletal muscle by both motor and sympathetic axons has been established, igniting interest in determining how the sympathetic nervous system (SNS) affect skeletal muscle composition and function throughout the lifetime. Selective expression of the heart and neural crest derivative 2 gene in peripheral SNs increases muscle mass and force regulating skeletal muscle sympathetic and motor innervation; improving acetylcholine receptor stability and NMJ transmission; preventing inflammation and myofibrillar protein degradation; increasing autophagy; and probably enhancing protein synthesis. Elucidating the role of central SNs will help to define the coordinated response of the visceral and neuromuscular system to physiological and pathological challenges across ages. This review discusses the following questions: (1) Does the SNS regulate skeletal muscle motor innervation? (2) Does the SNS regulate presynaptic and postsynaptic neuromuscular junction (NMJ) structure and function? (3) Does sympathetic neuron (SN) regulation of NMJ transmission decline with aging? (4) Does maintenance of SNs attenuate aging sarcopenia? and (5) Do central SN group relays influence sympathetic and motor muscle innervation?

Heart and neural crest derivative 2-induced preservation of sympathetic neurons attenuates sarcopenia with aging.

BACKGROUND: Sarcopenia, or age-dependent decline in muscle force and power, impairs mobility, increasing the risk of falls, institutionalization, co-morbidity, and premature death. The discovery of adrenoceptors, which mediate the effects of the sympathetic nervous system (SNS) neurotransmitter norepinephrine on specific tissues, sparked the development of sympathomimetics that have profound influence on skeletal muscle mass. However, chronic administration has serious side effects that preclude their use for muscle-wasting conditions. Interventions that can adjust neurotransmitter release to changing physiological demands depend on understanding how the SNS affects neuromuscular transmission, muscle motor innervation, and muscle mass. METHODS: We examined age-dependent expression of the heart and neural crest derivative 2 (Hand2), a critical transcription factor for SN maintenance, and we tested the possibility that inducing its expression exclusively in sympathetic neurons (SN) will prevent (i) motor denervation, (ii) impaired neuromuscular junction (NMJ) transmission, and (iii) loss of muscle mass and function in old mice. To test this hypothesis, we delivered a viral vector carrying Hand2 expression or an empty vector exclusively in SNs by vein injection in 16-month-old C57BL/6 mice that were sacrificed 6 months later. Techniques include RNA-sequencing, real-time PCR, genomic DNA methylation, viral vector construct, tissue immunohistochemistry, immunoblot, confocal microscopy, electrophysiology, and in vivo mouse physical performance. RESULTS: Hand2 expression declines throughout life, but inducing its expression increased (i) the number and size of SNs, (ii) muscle sympathetic innervation, (iii) muscle weight and force and whole-body strength, (iv) myofiber size but not muscle fibre-type composition, (v) NMJ transmission and nerve-evoked muscle force, and (vi) motor innervation in old mice. Additionally, the SN controls a set of genes to reduce inflammation and to promote transcription factor activity, cell signalling, and synapse in the skeletal muscle. Hand2 DNA methylation may contribute, at least partially, to gene silencing. CONCLUSIONS: Selective expression of Hand2 in the mouse SNs from middle age through old age increases muscle mass and force by (i) regulating skeletal muscle sympathetic and motor innervation; (ii) improving acetylcholine receptor stability and NMJ transmission; (iii) preventing inflammation and myofibrillar protein degradation; (iv) increasing autophagy; and (v) probably enhancing protein synthesis.

Aging Blunts Sympathetic Neuron Regulation of Motoneurons Synaptic Vesicle Release Mediated by ß1- and α2B-Adrenergic Receptors in Geriatric Mice.

This study was designed to determine whether and how the sympathetic nervous system (SNS) regulates motoneuron axon function and neuromuscular transmission in young (3-4-month) and geriatric (31-month) mice. Our approach included sciatic-peroneal nerve immunolabeling coregistration, and electrophysiological recordings in a novel mouse ex-vivo preparation, the sympathetic-peroneal nerve-lumbricalis muscle (SPNL). Here, the interaction between the motoneuron and SNS at the neuromuscular junction (NMJ) and muscle innervation reflect the complexity of the living mouse. Our data show that electrical stimulation of the sympathetic neuron at the paravertebral ganglia chain enhances motoneuron synaptic vesicle release at the NMJ in young mice, while in geriatric mice, this effect is blunted. We also found that blocking ß-AR prevents the sympathetic neuron from increasing NMJ transmission. Immunofluorescence coexpression analysis of immunolabeled ARs with choline acetyltransferase-, tyrosine hydroxylase-, or calcitonin gene-related peptide immunoreactive axons showed that α2B-AR is found mainly in sympathetic neurons, ß1-AR in sympathetic- and motor-neurons, and both decline significantly with aging. In summary, this study unveils the molecular substrate accounting for the influence of endogenous sympathetic neurons on motoneuron-muscle transmission in young mice and its decline with aging.

Sympathomimetics regulate neuromuscular junction transmission through TRPV1, P/Q- and N-type Ca2+ channels.

Increasing evidence indicates that, first, the sympathetic nervous system interacts extensively with both vasculature and skeletal muscle fibers near neuromuscular junctions (NMJs) and, second, its neurotransmitter, noradrenaline, influences myofiber molecular composition and function and motor innervation. Since sympathomimetic agents have been reported to improve NMJ transmission, we examined whether two in clinical use, salbutamol and clenbuterol, affect the motor axon terminal via extracellular Ca2+ and molecular targets, such as TRPV1 and P/Q- and N-type voltage-activated Ca2+ channels. Electrophysiological recordings in ex-vivo preparations of peroneal nerves and lumbricalis muscles from young adult mice focused on spontaneous miniature end-plate potentials and singly and repetitively evoked end-plate potentials. Adding one dose of salbutamol or clenbuterol to the nerve/muscle preparation or repeatedly administering salbutamol to a mouse for 4 weeks increased spontaneous and evoked synaptic vesicle release but induced a steep decline in EPP amplitude in response to repetitive nerve stimulation. These effects were mediated primarily by ω-agatoxin IVA-sensitive P/Q-type and secondarily by ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Presynaptic arvanil-sensitive TRPV1 channels seem to regulate Ca2+ at the motor neuron terminal at rest, while putative presynaptic ß-adrenergic receptors may mediate sympathomimetic and catecholamine effects on presynaptic Ca2+ channels during NMJ activation.

Relationship of Physical Function to Single Muscle Fiber Contractility in Older Adults: Effects of Resistance Training With and Without Caloric Restriction.

BACKGROUND: Previous studies support beneficial effects of both resistance exercise training (RT) and caloric restriction (CR) on skeletal muscle strength and physical performance. The goal of this study was to determine the effects of adding CR to RT on single-muscle fiber contractility responses to RT in older overweight and obese adults. METHODS: We analyzed contractile properties in 1,253 single myofiber from muscle biopsies of the vastus lateralis, as well as physical performance and thigh muscle volume, in 31 older (65-80 years), overweight or obese (body mass index = 27-35 kg/m2) men (n = 19) and women (n = 12) who were randomly assigned to a standardized, progressive RT intervention with CR (RT+CR; n = 15) or without CR (RT; n = 16) for 5 months. RESULTS: Both interventions evoked an increase in force normalized to cross-sectional area (CSA), in type-I and type-II fibers and knee extensor quality. However, these improvements were not different between intervention groups. In the RT group, changes in total thigh fat volume inversely correlated with changes in type-II fiber force (r = -.691; p = .019). Within the RT+CR group, changes in gait speed correlated positively with changes in type-I fiber CSA (r = .561; p = .030). In addition, increases in type-I normalized fiber force were related to decreases in thigh intermuscular fat volume (r = -0.539; p = .038). CONCLUSION: Single muscle fiber force and knee extensor quality improve with RT and RT+CR; however, CR does not enhance improvements in single muscle fiber contractility or whole muscle in response to RT in older overweight and obese men and women.

Troponin T3 associates with DNA consensus sequence that overlaps with p53 binding motifs.

We recently reported that in addition to its classical cytoplasmic location, the fast skeletal muscle Troponin T3 (TnT3) shuttles to the nucleus, where it appears to perform nonclassical transcription regulatory functions. Importantly, changes in the composition of the nucleus-localized pool of TnT3 and its fragments contribute to age-dependent muscle damage and wasting. Here, using ChIP-Seq, we demonstrate that TnT3 associates with DNA consensus sequences including the TGCCT motif, which is required for p53 binding to the promoter area of p53-related genes. Gene set enrichment analysis further demonstrated that the p53 pathway was the most significantly enriched pathway among genes annotated to the TnT3 ChIP-Seq peaks. We further demonstrated a strong correlation (r = 0.78, P = 1 × 10-4) between the expression levels of TNNT3 and TP53-inducible ribonucleotide reductase regulatory subunit M2B (RRM2B) in skeletal muscle tissue of 21 lean non-diabetic human subjects and a significant (P < 0.05) reduction in the levels of both gene transcripts in the third age-tertile group [42.3-70 years of age (yoa)] as compared to the second age-tertile (31.3-42.3 yoa). Of note, both TNNT3 and RRM2B expression levels negatively associated with total body fat mass (each with r = 0.49, P < 0.05), whereas RRM2B positively correlated with pancreatic ß cell function (rRRM2B~HOMA-B = 0.47, P = 0.047). This work suggests that reduced TNNT3 gene expression is another mechanism leading to reduced TnT3 and excitation-contraction coupling with aging. Consequently, TnT3 appears to contribute to age-related sarcopenia and possibly other age-related deficiencies such as muscle insulin resistance and ß cell dysfunction by interacting with TnT3-binding sequences in the promoter area of p53-related genes, among others, and consequently modulating the transcriptional regulation of these target genes.

Novel Peripherally Derived Neural-Like Stem Cells as Therapeutic Carriers for Treating Glioblastomas.

Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary malignant adult brain tumor characterized by extensive invasiveness, heterogeneity, and angiogenesis. Standard treatment options such as radiation and chemotherapy have proven to be only marginally effective in treating GBM because of its invasive nature. Therefore, extensive efforts have been put forth to develop tumor-tropic stem cells as viable therapeutic vehicles with potential to treat even the most invasive tumor cells that are harbored within areas of normal brain. To this end, we discovered a newly described NG2-expressing cell that we isolated from a distinct pericyte subtype found abundantly in cultures derived from peripheral muscle. In this work, we show the translational significance of these peripherally derived neural-like stem cells (NLSC) and their potential to migrate toward tumors and act as therapeutic carriers. We demonstrate that these NLSCs exhibit in vitro and in vivo GBM tropism. Furthermore, NLSCs did not promote angiogenesis or transform into tumor-associated stromal cells, which are concerns raised when using other common stem cells, such as mesenchymal stem cells and induced neural stem cells, as therapeutic carriers. We also demonstrate the potential of NLSCs to express a prototype therapeutic, tumor necrosis factor α-related apoptosis-inducing ligand and kill GBM cells in vitro. These data demonstrate the therapeutic potential of our newly characterized NLSC against GBM. Stem Cells Translational Medicine 2017;6:471-481.

BDA-410 Treatment Reduces Body Weight and Fat Content by Enhancing Lipolysis in Sedentary Senescent Mice.

Loss of muscle mass and force with age leads to fall risk, mobility impairment, and reduced quality of life. This article shows that BDA-410, a calpain inhibitor, induced loss of body weight and fat but not lean mass or skeletal muscle proteins in a cohort of sedentary 23-month-old mice. Food and water intake and locomotor activity were not modified, whereas BDA-410 treatment decreased intramyocellular lipid and perigonadal fat, increased serum nonesterified fatty acids, and upregulated the genes mediating lipolysis and oxidation, lean phenotype, muscle contraction, muscle transcription regulation, and oxidative stress response. This finding is consistent with our recent report that lipid accumulation in skeletal myofibers is significantly correlated with slower fiber-contraction kinetics and diminished power in obese older adult mice. A proteomic analysis and immunoblot showed downregulation of the phosphatase PPP1R12B, which increases phosphorylated myosin half-life and modulates the calcium sensitivity of the contractile apparatus. This study demonstrates that BDA-410 exerts a beneficial effect on skeletal muscle contractility through new, alternative mechanisms, including enhanced lipolysis, upregulation of "lean phenotype-related genes," downregulation of the PP1R12B phosphatase, and enhanced excitation-contraction coupling. This single compound holds promise for treating age-dependent decline in muscle composition and strength.

Resistance Training Enhances Skeletal Muscle Innervation Without Modifying the Number of Satellite Cells or their Myofiber Association in Obese Older Adults.

Studies in humans and animal models provide compelling evidence for age-related skeletal muscle denervation, which may contribute to muscle fiber atrophy and loss. Skeletal muscle denervation seems relentless; however, long-term, high-intensity physical activity appears to promote muscle reinnervation. Whether 5-month resistance training (RT) enhances skeletal muscle innervation in obese older adults is unknown. This study found that neural cell-adhesion molecule, NCAM+ muscle area decreased with RT and was inversely correlated with muscle strength. NCAM1 and RUNX1 gene transcripts significantly decreased with the intervention. Type I and type II fiber grouping in the vastus lateralis did not change significantly but increases in leg press and knee extensor strength inversely correlated with type I, but not with type II, fiber grouping. RT did not modify the total number of satellite cells, their number per area, or the number associated with specific fiber subtypes or innervated/denervated fibers. Our results suggest that RT has a beneficial impact on skeletal innervation, even when started late in life by sedentary obese older adults.

Troponin T3 regulates nuclear localization of the calcium channel Cavß1a subunit in skeletal muscle.

The voltage-gated calcium channel (Cav) ß1a subunit (Cavß1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavß1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavß1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavß1a/YFP shows that TnT3 facilitates Cavß1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.

Pericytes at the intersection between tissue regeneration and pathology.

Perivascular multipotent cells, pericytes, contribute to the generation and repair of various tissues in response to injury. They are heterogeneous in their morphology, distribution, origin and markers, and elucidating their molecular and cellular differences may inform novel treatments for disorders in which tissue regeneration is either impaired or excessive. Moreover, these discoveries offer novel cellular targets for therapeutic approaches to many diseases. This review discusses recent studies that support the concept that pericyte subtypes play a distinctive role in myogenesis, neurogenesis, adipogenesis, fibrogenesis and angiogenesis.

Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-dependent manner.

INTRODUCTION: Fibrosis, or scar formation, is a pathological condition characterized by excessive production and accumulation of collagen, loss of tissue architecture, and organ failure in response to uncontrolled wound healing. Several cellular populations have been implicated, including bone marrow-derived circulating fibrocytes, endothelial cells, resident fibroblasts, epithelial cells, and recently, perivascular cells called pericytes. We previously demonstrated pericyte functional heterogeneity in skeletal muscle. Whether pericyte subtypes are present in other tissues and whether a specific pericyte subset contributes to organ fibrosis are unknown. METHODS: Here, we report the presence of two pericyte subtypes, type-1 (Nestin-GFP-/NG2-DsRed+) and type-2 (Nestin-GFP+/NG2-DsRed+), surrounding blood vessels in lungs, kidneys, heart, spinal cord, and brain. Using Nestin-GFP/NG2-DsRed transgenic mice, we induced pulmonary, renal, cardiac, spinal cord, and cortical injuries to investigate the contributions of pericyte subtypes to fibrous tissue formation in vivo. RESULTS: A fraction of the lung's collagen-producing cells corresponds to type-1 pericytes and kidney and heart pericytes do not produce collagen in pathological fibrosis. Note that type-1, but not type-2, pericytes increase and accumulate near the fibrotic tissue in all organs analyzed. Surprisingly, after CNS injury, type-1 pericytes differ from scar-forming PDGFRß + cells. CONCLUSIONS: Pericyte subpopulations respond differentially to tissue injury, and the production of collagen by type-1 pericytes is organ-dependent. Characterization of the mechanisms underlying scar formation generates cellular targets for future anti-fibrotic therapeutics.

The Cavß1a subunit regulates gene expression and suppresses myogenin in muscle progenitor cells.

Voltage-gated calcium channel (Cav) ß subunits are auxiliary subunits to Cavs. Recent reports show Cavß subunits may enter the nucleus and suggest a role in transcriptional regulation, but the physiological relevance of this localization remains unclear. We sought to define the nuclear function of Cavß in muscle progenitor cells (MPCs). We found that Cavß1a is expressed in proliferating MPCs, before expression of the calcium conducting subunit Cav1.1, and enters the nucleus. Loss of Cavß1a expression impaired MPC expansion in vitro and in vivo and caused widespread changes in global gene expression, including up-regulation of myogenin. Additionally, we found that Cavß1a localizes to the promoter region of a number of genes, preferentially at noncanonical (NC) E-box sites. Cavß1a binds to a region of the Myog promoter containing an NC E-box, suggesting a mechanism for inhibition of myogenin gene expression. This work indicates that Cavß1a acts as a Cav-independent regulator of gene expression in MPCs, and is required for their normal expansion during myogenic development.

Type-2 pericytes participate in normal and tumoral angiogenesis.

Tissue growth and function depend on vascularization, and vascular insufficiency or excess exacerbates many human diseases. Identification of the biological processes involved in angiogenesis will dictate strategies to modulate reduced or excessive vessel formation. We examine the essential role of pericytes. Their heterogeneous morphology, distribution, origins, and physiology have been described. Using double-transgenic Nestin-GFP/NG2-DsRed mice, we identified two pericyte subsets. We found that Nestin-GFP(-)/NG2-DsRed(+) (type-1) and Nestin-GFP(+)/NG2-DsRed(+) (type-2) pericytes attach to the walls of small and large blood vessels in vivo; in vitro, type-2, but not type-1, pericytes spark endothelial cells to form new vessels. Matrigel assay showed that only type-2 pericytes participate in normal angiogenesis. Moreover, when cancer cells were transplanted into Nestin-GFP/NG2-DsRed mice, type-1 pericytes did not penetrate the tumor, while type-2 pericytes were recruited during its angiogenesis. As inhibition of angiogenesis is a promising strategy in cancer therapy, type-2 pericytes may provide a cellular target susceptible to signaling and pharmacological manipulation in treating malignancy. This work also reports the potential of type-2 pericytes to improve blood perfusion in ischemic hindlimbs, indicating their potential for treating ischemic illnesses.

Type-1 pericytes participate in fibrous tissue deposition in aged skeletal muscle.

In older adults, changes in skeletal muscle composition are associated with increased fibrosis, loss of mass, and decreased force, which can lead to dependency, morbidity, and mortality. Understanding the biological mechanisms responsible is essential to sustaining and improving their quality of life. Compared with young mice, aged mice take longer to recover from muscle injury; their tissue fibrosis is more extensive, and regenerated myofibers are smaller. Strong evidence indicates that cells called pericytes, embedded in the basement membrane of capillaries, contribute to the satellite-cell pool and muscle growth. In addition to their role in skeletal muscle repair, after tissue damage, they detach from capillaries and migrate to the interstitial space to participate in fibrosis formation. Here we distinguish two bona fide pericyte subtypes in the skeletal muscle interstitium, type-1 (Nestin-GFP(-)/NG2-DsRed(+)) and type-2 (Nestin-GFP(+)/NG2-DsRed(+)), and characterize their heretofore unknown specific roles in the aging environment. Our in vitro results show that type-1 and type-2 pericytes are either fibrogenic or myogenic, respectively. Transplantation studies in young animals indicate that type-2 pericytes are myogenic, while type-1 pericytes remain in the interstitial space. In older mice, however, the muscular regenerative capacity of type-2 pericytes is limited, and type-1 pericytes produce collagen, contributing to fibrous tissue deposition. We conclude that in injured muscles from aging mice, the pericytes involved in skeletal muscle repair differ from those associated with scar formation.