Metformin shown for the first time to improve behavior in humans with Fragile X Mental Retardation Syndrome: AMPK links Progeria, FXS, & Down syndrome

By Vanellus Foto (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons; "Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; By Peter Saxon (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons

A study recently published in the journal Clinical Genetics in April of 2017 by researchers from the University of California, Davis Medical Center and the University of Colorado School of Medicine demonstrated for the first time that metformin consistently improved behavior in several patients diagnosed with Fragile X Syndrome (FXS), a genetic disorder characterized by intellectual disability and significant deficits in neurological function and cognitive development [1]. An improvement in behavior was documented in the Aberrant Behavior Checklist (ABC) for all cases, as evidenced by consistent improvements (i.e. lower scores compared to pre-metformin treatment) in social avoidance, irritability, hyperactivity, and social unresponsiveness (see table below) as well as improvements in language and conversational skills reported by familial caretakers [1]. Interestingly, a study published in 2016 showed that metformin rescued and restored memory deficits in a Drosophila (fly) model of FXS and a soon-to-be published study currently in press also demonstrated that metformin restored behavioral and morphological defects in a mouse model of FXS [2-4].

In 2014 and 2015, I was first to hypothesize and publish that AMPK activation via compounds including metformin would beneficially alter gene splicing and alleviate accelerated aging defects in cells derived from patients with the genetic disorder Hutchinson-Gilford progeria syndrome, which was later substantiated by studies published in 2016 and 2017 (see below). Because AMPK activation has been shown to restore severe mitochondrial impairment in hippocampal progenitor cells from a Down syndrome mouse model and metformin reverses mitochondrial dysfunction in human fetal cells with Down syndrome, the activation of AMPK via the induction of cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links Fragile X syndrome, Hutchison-Gilford progeria syndrome, and Down syndrome with the therapeutic effects of structurally dissimilar AMPK activators including metformin, resveratrol, and lithium. Indeed, in my recently submitted manuscripts and publications, I initially propose that all three of the aforementioned genetic disorders as well as learning and memory (i.e. hippocampal long-term potentiation), adult and cancer stem cells, and the creation all human life (via oocyte activation/sperm acrosome reaction) are connected to HIV-1 latency via AMPK activation [5-10].

Fragile X syndrome (FXS) is the most common inherited cause of autism spectrum disorder (ASD) and intellectual disability (ID) and results from a trinucelotide expansion-induced hypermethylation and silencing of the Fragile X Mental Retardation 1 gene (FMR1) and resultant silencing of the FMR1 gene product FMR1 protein (FMRP) [1]. Symptomatic behaviors associated with FXS include aggression, self-injurious behaviors, anxiety, and attention deficit hyperactivity disorder (ADHD). In the study, several patients, listed below, were treated clinically with metformin at the Fragile X Treatment and Research Center at the University of California, Davis Medical Center MIND Institute for at least six months along with assessments of medical and behavioral co-morbidities and laboratory studies before and after treatment [1].

Case 1:

Case 1 was a 19 year-old male diagnosed with FXS and ASD at age 5 with a history of hyperphagia, characteristic features of FXS including macroorchidism (abnormally large testicles), and behavioral issues including aggression, hand biting, hyperactivity, ADHD, poor eye contact, and skin picking and scratching [1]. After being placed on several mediations (e.g. minocycline, divalproex, etc.), he was started on metformin 500 mg twice a day (bid) and subsequently experienced significant improvements in hyperphagia, irritability, aggression, communication, and behavior. It was also noted that the patient no longer focuses on food (as he did for many years), now enjoys outdoor activities, and has considerably improved self-esteem [1].    

Case 2:

Case 2 was a 13 year-old boy with FXS who at age 10 had a Leiter IQ of 44 and an Autism Diagnostic Observation Schedule (ADOS) total score of 25 (well into ASD range) [1]. Behavioral symptoms consisted of hyperphagia, severe hyperactivity and impulsivity, significant aggression characterized by kicking and hitting at home and at school, and escalating aggressiveness towards his mother (which led to her becoming depressed), leading to his placement in a residential group home to control his eating and aggression [1]. After starting metformin 500 mg bid at age 12, his mother and care center noted significant improvements in his behavior over the course of a year, including the patient being more calm and patient, negotiating with teachers more, displaying a better understanding when things were explained to him, being more responsive to rewards, and able to follow directions better and work longer with less agitation and aggression [1].

Case 3:

Case 3 was another 19-year-old boy diagnosed at age 3 with FXS and had also been diagnosed with ADHD, ASD, and specific phobias [1].  Behavioral symptoms included impulsive episodic outbursts, persistent hyperphagia, skin pricking, enuresis (inability to control urination), and binge eating to the point of vomiting. After starting metformin 500 mg bid, the patient experienced significant behavioral improvements including a decrease in tantrums, improvement in communication ability, and cessation of head-banging and other self-injurious behavior [1].

Case 4

Case 4 was a 60-year-old female diagnosed with FXS and mild ID currently residing in a group home with a history of morbid obesity along with repetitive behavior, memory deficits, agitation, and anxiety that caused concern among her caregivers [1]. After starting on metformin 500 mg bid, the patient is no longer overeating, has lost 41.3 pounds, and her irritability and social responsiveness has improved (see table below) [1].

Case 5 

Case 5 was a 31-year-old man diagnosed with FXS and ASD with a full scale IQ of 54 on the WISC III. The patient exhibited macroorchidism and also had problems with anxiety and tantrums [1]. After starting metformin 500 mg bid, the patient’s appetite decreased, more language was noted by his speech and language therapist, and he has improved self-initiative. Also, he “will carry out chores such as cleaning his room or cleaning the house without excessive urging or reminders, helps his mom with gardening and is able to coordinate the rake better, has a lot of trivia knowledge, and asks more inquisitive questions. The mother is able to have a discussion with him even about emotional issues, he is able to talk about his grief and to relate his father's death to his excessive smoking and drinking problems, and he seems to be putting together concepts better and having back-and-forth conversations.” [1].

Case 6

Case 6 was originally seen at 10 years of age and exhibited typical characteristics of FXS on physical examination, including soft skin, flat feet, and poor eye contact [1]. DNA testing confirmed a diagnosis of FXS and the patient demonstrated a full scale IQ of 50. The patient also displayed a dramatic increase in anxiety, aversion to people, and intermittent aggression and headaches due to a traumatic episode wherein the patient had to be tasered by police at a hospital due to his aggression [1].  After starting metformin 500 mg bid at age 24, he has not experienced on outburst, “seems happier and more active, has improvement in language, is able to carry out a two way conversation, has become more outgoing socially with less anxiety, and eats without gorging himself.” [1].

Case 7

Case 7 was a 4 year old boy diagnosed with FXS at 14 months and displayed features typical of FXS, including prominent ears, a broad forehead, and flat feet [1]. He had motor and language delays at an early age and the parents self-treated the child with a cannabinoid tincture at age 2 due to staring spells and a lack of language. The parents noted that the tincture led to a decrease in staring spells, increased verbalization, and improved anxiety [1]. “After 6 months of a dose of metformin 50 mg the family felt he has fewer outbursts or tantrums, better attention, less hyperactivity and improvements in his language. Developmental testing after 4 months of treatment at 4.5 years showed expressive language at the 31 month age equivalent.” [1]. 

As demonstrated by these case studies, it appears that metformin significantly improves cognitive and behavioral deficits in the genetic disorder FXS. Additionally, as noted above, metformin has recently been shown to rescue and restore memory deficits in a Drosophila model of FXS and a study in press has shown that metformin corrected social novelty impairment, reduced testicular weight, decreased repetitive grooming, rescued excessive long-term depression and dendritic spine abnormalities, and altered excitatory synaptic transmission in an FXS mouse model, indicating that metformin likely exerts significant beneficial effects in disparate genetic disorders [2-4]. Indeed, metformin has also been shown to correct alternative splicing defects in primary myoblasts derived from patients with myotonic dystrophy type I (DM1) as well as in derivatives of embryonic stem cells that carry the DM1 mutation via activation of AMPK. DM1 is a genetic disorder characterized by muscle wasting that is also caused by the expansion of a trinucleotide repeat, similar to FXS [11]. As further explained below, because metformin activates AMPK and alleviates accelerated aging defects in cells from Hutchinson-Gilford progeria syndrome (HGPS) patients and because metformin also rescues mitochondrial defects in human fetal cells with Down syndrome (DS), AMPK activation induced by cellular stress (i.e. AMP/ATP ratio increase, reactive oxygen species generation, and/or intracellular calcium increases, etc.) links the amelioration and/or reversal of pathological cellular defects in FXS, HGPS, DM1, and DS with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation all human life (via oocyte activation/sperm acrosome reaction), hypotheses that I first proposed in several publications and pending manuscripts [5-10].

Down syndrome (DS) is caused by either a partial or full trisomy of chromosome 21 and is associated with impairments in cognition, learning and memory, and disorders of the immune system [12]. Human and animal studies strongly suggest that mitochondrial dysfunction is associated with pathogenic features of DS and mitochondrial abnormalities have been found in all DS cells analyzed in culture to date, including neurons, astrocytes, and lymphocytes [12]. Indeed, in DS human fetal fibroblasts, PGC-1α, a master regulator of mitochondrial biogenesis, is down regulated at both the mRNA and protein levels and AMPK phosphorylation of the PGC-1α protein is essential for PGC-1α-dependent induction of the PGC-1α promoter [12].

Izzo et al. initially observed that DS human fetal fibroblasts (DS-HFFs) displayed a 40%-50% reduction in PGC-1α mRNA and protein levels compared to non-trisomic counterparts (N-HFFs). DS-HFFs treated with metformin however led to a significant increase in PGC-1α at both the mRNA and protein levels compared to untreated control cells, an increase in the mRNA levels of NRF-1 and TFAM (PGC-1α target genes critical in promoting mitochondrial biogenesis), and an increase in mtDNA content [12]. Additionally, compared to N-HFFs, DS-HFFs were characterized by a decrease in basal oxygen consumption rate (OCR, ~55% inhibition), a decrease in OCR related to ATP production (~58% inhibition), and a reduction in maximal respiratory capacity (~58% inhibition). DS-HFFs treated with metformin increased basal OCR, OCR related to ATP production, ATP concentration, mitochondrial membrane potential, and maximal respiratory capacity, thus restoring mitochondrial respiration in DS-HFFs [12].

Furthermore, compared to N-HFFs, DS-HFFs exhibited extensive mitochondrial damage, as evidenced by swollen cristae (~60%-90% of DS-HFFs vs. ~15% of N-HFFs), mitochondria with intra-oedema (~40% of DS-HFFs vs. ~6% of N-HFFs), and an increase in the number of damaged mitochondria (~90% in DS-HFFs vs. ~25% in N-HFFs). Metformin-treated DS-HFFs, compared to untreated cells, displayed narrower cristae with widths that were comparable to N-HFFs cells, fewer damaged mitochondria (~40%), and fewer mitochondria with intra-oedema (~5%) [12]. Interestingly, as mitochondrial fusion also plays an important role in mitochondrial functionality, the mRNA and protein expression of OPA1 and MFN2 (genes that regulate mitochondrial fusion) were significantly down-regulated in DS-HFFs vs. N-HFFs and correlated with extensive mitochondrial fragmentation in DS-HFFs. Treatment of DS-HFFs with metformin significantly increased the mRNA and protein levels of OPA1 and MFN2 and also reduced fragmentation of the mitochondrial network, indicating that metformin induces correction of mitochondrial phenotype by also restoring mitochondrial fusion machinery [12].      

The AMPK activator EGCG also reverses cognitive defects in DS patients, promotes oxidative phosphorylation and mitochondrial biogenesis in lymphoblasts and fibroblasts from DS patients (i.e. increased levels and activity of PGC-1α, NRF-1 and TFAM), and restores oxidative phosphorylation, mitochondrial biogenesis, and improves hippocampal neural progenitor cell proliferation in a DS mouse model (Ts65Dn) in combination with the polyphenol resveratrol (i.e. increase in PGC-1α) in an AMPK-dependent manner [13-15]. Mitochondrial dysfunction is also a prominent feature associated with the accelerated aging disorder Hutchison-Gilford progeria syndrome (HGPS) and metformin has recently been shown to alleviate accelerated aging defects, activate AMPK, and beneficially alter gene splicing in HGPS patient cells.

Egesipe et al. initially demonstrated, using mesenchymal stem cells (MSCs) derived from HGPS induced pluripotent stem cells (i.e. HGPS MSCs), a significant dose-dependent decrease in SRSF1 mRNA levels after metformin treatment and up to a 40% decrease in SRSF1 protein levels after treatment with 5 mmol/l of metformin (SRSF1 is a gene splicing factor that is upregulated in HGPS cells and causes faulty gene splicing of the LMNA gene leading to increased production of the toxic protein progerin) [16]. A significant decrease was also observed in both lamin A and progerin mRNA expression in HGPS MSCs treated with 5 mmol/l metformin, with progerin mRNA expression and protein levels reduced to levels lower than that of lamin A mRNA expression and protein levels, indicating that metformin-induced inhibition of SRSF1 led to an increase in the lamin A/progerin ratio and thus beneficially altered gene splicing [16].       

The results obtained using HGPS MSCs were also replicated in additional in vitro cell models, with 5 mmol/l of metformin decreasing progerin mRNA expression up to 50% in LmnaG609G/G609G mouse primary fibroblasts (HGPS mouse model) and decreasing both lamin A and progerin mRNA expression in primary HGPS fibroblasts [16]. Interestingly, 5 mmol/l of metformin also decreased progerin mRNA expression in wild-type/normal MSCs that had been incubated with a compound that induces progerin expression, indicating that metformin may also prove beneficial in reducing progerin levels in normal humans [16].

Most importantly, however, treatment of HGPS MSCs with 5 mmol/l of metformin reduced the percentage of abnormal nuclei from 60% pre-treatment to less than 40% after treatment (wild-type MSCs presented less than 20% of abnormal nuclei). The metformin-induced reduction in abnormal nuclei was comparable to the reference treatment tipifarnib (1 μmol/1), a farnesyl-transferase inhibitor [16]. Additionally, as HGPS MSCs are characterized by premature osteogenic differentiation (indicated by increased alkaline phosphatase activity compared to wild-type osteogenic progenitor cells), 5 mmol/l of metformin led to a significant rescue of alkaline phosphatase activity in HGPS osteogenic progenitor cells, comparable to levels found in tipifarnib-treated cells [16].

Park et al. also characterized the cellular phenotypes of primary dermal fibroblasts derived from HGPS patients of different ages and showed increased staining of senescence-associated beta-galactosidase (SA-β-gal, an indicator of cellular senescence) and increased levels of mitochondrial superoxide in HG8 cells (from an 8 year old patient) compared to HG3 (3 year old) and HG5 (5 year old) cells [17]. All cells expressed the toxic protein progerin, superoxide dismutase 2 (SOD2, a mitochondrial antioxidant enzyme) was highest in normal fibroblasts and lowest in HG8 cells, and cellular proliferation rate slowed at an earlier time in HGPS cells compared to normal fibroblasts [17].

Utilizing HG8 cells (which demonstrated the highest levels of senescence), the authors also elucidated the effects of metformin (2mM), rapamycin (200nM), or a combination of both drugs on the nuclear phenotype of HGPS cells. Rapamycin significantly decreased the number of nuclei with abnormal morphology and metformin treatment also led to a significant increase in the number of cells with normal nuclei compared to control-treated cells [17]. Metformin also reduced senescence in HGPS cells (i.e. reduction in SA-β-gal staining) and co-treatment with rapamycin and metformin led to an approximately 34.2 % inhibition of senescence, with similar results observed in HG3 and HG8 cells [3]. Metformin, rapamycin, or co-treatment with both compounds led to a significant reduction in the number of cells containing more than 20 γ-H2AX foci (a marker of DNA damage) in HG8, HG3, and HG5 cells, indicating that metformin increases the efficiency of DNA repair in HGPS cells [17].

Metformin also exerted antioxidant effects in HGPS cells, as evidenced by a significant decrease in ROS production and mitochondrial superoxide formation compared to control cells as well as an upregulation of SOD2 mRNA expression in aged BALB/c mice (>18 months old) [17]. Most importantly, metformin treatment at 2 and 20mM reduced progerin protein expression by approximately 20 % and 60 %, respectively, compared to mock-treated cells and increased the presence of normal nuclear phenotypes in HGPS cells [17]. Metformin treatment also significantly increased the phosphorylation and activation of AMPK in HGPS cells. Furthermore, western blot analysis indicated that rapamycin increased AMPK activation as well.

Strikingly, metformin has recently been shown to inhibit dengue virus replication in human liver cells in an AMPK-dependent manner, resveratrol (an AMPK activator) has recently been shown to reactivate latent HIV-1, and HIV-1 replication is significantly inhibited via knockdown of AMPK, indicating that AMPK activation leads to both the reactivation of latent viruses (facilitating immune system detection and destruction) as well as inhibition of productive viral replication [18-20]. AMPK activation also promotes oocyte meiotic induction and maturation (processes that are critical for efficient oocyte activation) and AMPK has recently been found localized across the entire acrosome in human spermatozoa [7,21,22]. The induction of cellular stress (e.g. increases in ROS, intracellular calcium, and/or AMP/ATP ratio increase), which activates AMPK, also promotes oocyte meiotic induction/maturation, oocyte activation, and the acrosome reaction in human sperm, processes critical for the creation of all human life [21,23,24]. Indeed, the calcium ionophore ionomycin, which activates AMPK, is commonly used to promote latent HIV-1 reactivation and is extensively used to activate human oocytes, creating normal healthy children [24-26]. Such evidence indicates and further substantiates the novel and provocative assertion that AMPK activation links the amelioration of pathological cellular defects in FXS, HGPS, DS, and DM1 with HIV-1 latency, adult and cancer stem cells, learning and memory, and the creation of all human life [5-10].

https://www.linkedin.com/pulse/metformin-shown-first-time-improve-behavior-humans-fragile-finley

*Table 2 adapted from: Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as Targeted Treatment in Fragile X Syndrome. Clin Genet. 2017 Apr 24. doi: 10.1111/cge.13039. [Epub ahead of print].

References

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