SMN protein is required throughout life to prevent spinal muscular atrophy disease progression.

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.

Pharmacologically induced mouse model of adult spinal muscular atrophy to evaluate effectiveness of therapeutics after disease onset.

Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.

Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.

Insulinotropic treatments exacerbate metabolic syndrome in mice lacking MeCP2 function.

Rett syndrome (RTT), an X-linked postnatal disorder, results from mutations in Methyl CpG-binding protein 2 (MECP2). Survival and breathing in Mecp2(NULL/Y) animals are improved by an N-terminal tripeptide of insulin-like growth factor I (IGF-I) treatment. We determined that Mecp2(NULL/Y) animals also have a metabolic syndrome and investigated whether IGF-I treatment might improve this phenotype. Mecp2(NULL/Y) mice were treated with a full-length IGF-I modified with the addition of polyethylene glycol (PEG-IGF-I), which improves pharmacological properties. Low-dose PEG-IGF-I treatment slightly improved lifespan and heart rate in Mecp2(NULL/Y) mice; however, high-dose PEG-IGF-I decreased lifespan. To determine whether insulinotropic off-target effects of PEG-IGF-I caused the detrimental effect, we treated Mecp2(NULL/Y) mice with insulin, which also decreased lifespan. Thus, the clinical benefit of IGF-I treatment in RTT may critically depend on the dose used, and caution should be taken when initiating clinical trials with these compounds because the beneficial therapeutic window is narrow.

Polyethylene glycol-coupled IGF1 delays motor function defects in a mouse model of spinal muscular atrophy with respiratory distress type 1.

Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd(2J)) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd(2J) mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd(2J) mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd(2J) mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.

Molecular, physiological, and motor performance defects in DMSXL mice carrying >1,000 CTG repeats from the human DM1 locus.

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes.

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I.

BACKGROUND: The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some of these functions involve regulation by the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, the functions and exact nature of these interactions remain incomplete. METHODS: IGF-I variants PEGylated at lysines K27, K65 and K68, were assessed for binding to IGFBPs using BIAcore, and for phosphorylation of the IGF-IR. Furthermore, functional consequences of PEGylation were investigated using cell viability and migration assays. In addition, downstream signaling pathways were analyzed using phospho-AKT and phospho-ERK1/2 assays. RESULTS: IGF-I PEGylated at lysines 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) was employed. Receptor phosphorylation was similarly reduced 2-fold with PEG-K65 and PEG-K68 in 3T3 fibroblasts and MCF-7 breast cancer cells, whereas PEG-K27 showed a more than 10- and 3-fold lower activation for 3T3 and MCF-7 cells, respectively. In addition, all PEG-IGF-I variants had a 10-fold reduced association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes, whereas cell viability was fully preserved. Analysis of downstream signaling revealed that AKT was preferentially affected upon treatment with PEG-IGF-I variants whereas MAPK signaling was unaffected by PEGylation. CONCLUSION: PEGylation of IGF-I has an impact on cell migration but not on cell viability. GENERAL SIGNIFICANCE: PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on receptor interaction as well as key extracellular proteins such as VN and IGFBPs.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Brain expression profiles of two SCN1A antisense RNAs in children and adolescents with epilepsy.

Objective: Heterozygous mutations within the voltage-gated sodium channel α subunit (SCN1A) are responsible for the majority of cases of Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. Development of novel therapeutic approaches is mandatory in order to directly target the molecular consequences of the genetic defect. The aim of the present study was to investigate whether cis-acting long non-coding RNAs (lncRNAs) of SCN1A are expressed in brain specimens of children and adolescent with epilepsy as these molecules comprise possible targets for precision-based therapy approaches. Methods: We investigated SCN1A mRNA expression and expression of two SCN1A related antisense RNAs in brain tissues in different age groups of pediatric non-Dravet patients who underwent surgery for drug resistant epilepsy. The effect of different antisense oligonucleotides (ASOs) directed against SCN1A specific antisense RNAs on SCN1A expression was tested. Results: The SCN1A related antisense RNAs SCN1A-dsAS (downstream antisense, RefSeq identifier: NR_110598) and SCN1A-usAS (upstream AS, SCN1A-AS, RefSeq identifier: NR_110260) were widely expressed in the brain of pediatric patients. Expression patterns revealed a negative correlation of SCN1A-dsAS and a positive correlation of lncRNA SCN1A-usAS with SCN1A mRNA expression. Transfection of SK-N-AS cells with an ASO targeted against SCN1A-dsAS was associated with a significant enhancement of SCN1A mRNA expression and reduction in SCN1A-dsAS transcripts. Conclusion: These findings support the role of SCN1A-dsAS in the suppression of SCN1A mRNA generation. Considering the haploinsufficiency in genetic SCN1A related DS, SCN1A-dsAS is an interesting target candidate for the development of ASOs (AntagoNATs) based precision medicine therapeutic approaches aiming to enhance SCN1A expression in DS.

Local application of engineered insulin-like growth factor I mRNA demonstrates regenerative therapeutic potential in vivo.

Insulin-like growth factor I (IGF-I) is a growth-promoting anabolic hormone that fosters cell growth and tissue homeostasis. IGF-I deficiency is associated with several diseases, including growth disorders and neurological and musculoskeletal diseases due to impaired regeneration. Despite the vast regenerative potential of IGF-I, its unfavorable pharmacokinetic profile has prevented it from being used therapeutically. In this study, we resolved these challenges by the local administration of IGF-I mRNA, which ensures desirable homeostatic kinetics and non-systemic, local dose-dependent expression of IGF-I protein. Furthermore, IGF-I mRNA constructs were sequence engineered with heterologous signal peptides, which improved in vitro protein secretion (2- to 6-fold) and accelerated in vivo functional regeneration (16-fold) over endogenous IGF-I mRNA. The regenerative potential of engineered IGF-I mRNA was validated in a mouse myotoxic muscle injury and rabbit spinal disc herniation models. Engineered IGF-I mRNA had a half-life of 17-25 h in muscle tissue and showed dose-dependent expression of IGF-I over 2-3 days. Animal models confirm that locally administered IGF-I mRNA remained at the site of injection, contributing to the safety profile of mRNA-based treatment in regenerative medicine. In summary, we demonstrate that engineered IGF-I mRNA holds therapeutic potential with high clinical translatability in different diseases.

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I).

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

Selective degeneration of septal and hippocampal GABAergic neurons in a mouse model of amyloidosis and tauopathy.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain accumulation of amyloid-ß peptide and neurofibrillary tangles, which are believed to initiate a pathological cascade that results in progressive impairment of cognitive functions and eventual neuronal death. To obtain a mouse model displaying the typical AD histopathology of amyloidosis and tauopathy, we generated a triple-transgenic mouse line (TauPS2APP) by overexpressing human mutations of the amyloid precursor protein, presenilin2 and tau genes. Stereological analysis of TauPS2APP mice revealed significant neurodegeneration of GABAergic septo-hippocampal projection neurons as well as their target cells, the GABAergic hippocampal interneurons. In contrast, the cholinergic medial septum neurons remained unaffected. Moreover, the degeneration of hippocampal GABAergic interneurons was dependent on the hippocampal subfield and interneuronal subtype investigated, whereby the dentate gyrus and the NPY-positive interneurons, respectively, were most strongly affected. Neurodegeneration was also accompanied by a change in the mRNA expression of markers for inhibitory interneurons. In line with the loss of inhibitory neurons, we observed functional changes in TauPS2APP mice relative to WT mice, with strongly enhanced long-term potentiation in the medial-perforant pathway input to the dentate gyrus, and stereotypic hyperactivity. Our data indicate that inhibitory neurons are the targets of neurodegeneration in a mouse model of amyloidosis and tauopathy, thus pointing to a possible role of the inhibitory network in the pathophysiological and functional cascade of Alzheimer's disease.

Functional improvement in mouse models of familial amyotrophic lateral sclerosis by PEGylated insulin-like growth factor I treatment depends on disease severity.

Insulin-like growth factor I (IGF-I) has been successfully tested in the SOD1-G93A mouse model of familial amyotrophic lateral sclerosis (ALS) and proposed for clinical treatment. However, beneficial effects required gene therapy or intrathecal application. Circumventing the dosing issues we recently found that polyethylene glycol (PEG) modified IGF-I (PEG-IGF-I) modulated neuromuscular function after systemic application, and protected against disease progression in a motor neuron disease model. Here we investigated its effects in two SOD1-G93A mouse lines, the G1L with a milder and the G1H with a more severe phenotype. Results showed that in G1L mice, PEG-IGF-I treatment significantly improved muscle force, motor coordination and animal survival. In contrast, treatment of G1H mice with PEG-IGF-I or IGF-I even at high doses did not beneficially affect survival or functional outcomes despite increased signalling in brain and spinal cord by both agents. In conclusion, the data point towards further investigation of the therapeutic potential of PEG-IGF-I in ALS patients with less severe clinical phenotypes.

Enhanced dentate gyrus synaptic plasticity but reduced neurogenesis in a mouse model of amyloidosis.

Long-term potentiation (LTP) and neurogenesis in the dentate gyrus (DG) are correlated forms of hippocampal plasticity which share, under physiological conditions, common regulatory mechanisms. In Alzheimer's disease (AD), their alterations are potentially associated with the early cellular pathology and cognitive decline. We analyzed DG LTP and neurogenesis in B6.152H mice, an amyloid precursor protein and presenilin 2 double-transgenic mouse model of amyloidosis and observed that DG LTP was strongly enhanced before and after amyloid plaque formation. Whereas proliferation of DG neuronal progenitor cells was unchanged, survival of newborn neurons was strongly decreased already before plaque formation. As similar alteration of neurogenesis was observed in PS2APP mice without changes in DG LTP (Richards et al. 2003), this study suggests that enhanced synaptic plasticity did not rescue impaired neurogenesis, and supports decreased survival of newborn neurons as an early event associated with AD detectable even before plaque formation.

Overlap of Peak Growth Activity and Peak IGF-1 to IGFBP Ratio: Delayed Increase of IGFBPs versus IGF-1 in Serum as a Mechanism to Speed up and down Postnatal Weight Gain in Mice.

Forced expression of insulin-like growth factor binding proteins (IGFBPs) in transgenic mice has clearly revealed inhibitory effects on somatic growth. However, by this approach, it cannot be solved if or how IGFBPs rule insulin-like growth factor (IGF)-dependent growth under normal conditions. In order to address this question, we have used growth-selected mouse models (obese and lean) and studied IGF-1 and IGFBPs in serum with respect to longitudinal growth activity in males and females compared with unselected controls. In mice of both genders, body weights were recorded and daily weight gains were calculated. Between 2 and 54 weeks of age, serum IGF-1 was determined by ELISA and intact IGFBP-2, -3 and -4 were quantified by Western ligand blotting. The molar ratio of IGF-1 to the sum of IGFBP-2 to -4 was calculated for all groups and plotted against the daily weight gain curve. Growth-selected mice are characterized by higher daily weight gains and extended periods of elevated growth activity if compared to matched unselected controls. Therefore, adult mice from the obese and lean groups can achieve more than twofold increased body weight in both genders (p < 0.001). Between 2 and 11 weeks of age, in obese and lean mice of both genders, serum IGF-1 concentrations are increased more prominently if compared to unselected controls (p < 0.001). Instead, substantial decreases of IGFBPs, particularly of IGFBP-2, are observed in males and females of all groups at the age of 2 to 4 weeks (p < 0.001). Due to the strong increase of IGF-1 but not of IGFBPs between two and four weeks of age, the ratio of IGF-1 to IGFBP-2 to -4 in serum significantly increased in all groups and genders (p < 0.05). Notably, the IGF-1 to IGFBP ratio was higher in male and female obese mice if compared to unselected controls (p < 0.05).

Cortical hypoperfusion in the B6.PS2APP mouse model for Alzheimer's disease: comprehensive phenotyping of vascular and tissular parameters by MRI.

Function and morphology of the cerebral vasculature were studied in the amyloid (Abeta) plaque-containing double-transgenic (TG) B6.PS2APP Alzheimer's disease (AD) mouse model with MRI at an age range of 10 to 17 months. Perfusion, blood volume, and average vessel geometry were assessed in the brain and compared to age-matched controls (wild-type [WT] C57Bl/6). Additionally, the MR relaxation times T(1), T(2), and T(2)* were measured to detect potential pathological changes that might be associated with Abeta plaque depositions. Both decreased perfusion and decreased blood volume were observed in the occipital cortex in B6.PS2APP mice as compared to controls. A significant decrease in T(1) and T(2) was found in the frontal cortex and in the subiculum/parasubiculum. Immunohistochemistry confirmed plaque depositions in the cortex and in the subiculum/parasubiculum. In summary, our data indicate a reduced blood supply of B6.PS2APP mice in the occipital cortex that parallels the findings in cortical regions of patients with AD.

Nerve sprouting capacity in a pharmacologically induced mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.

Disturbed cross talk between insulin-like growth factor I and AMP-activated protein kinase as a possible cause of vascular dysfunction in the amyloid precursor protein/presenilin 2 mouse model of Alzheimer's disease.

Cerebrovascular dysfunction appears to be involved in Alzheimer's disease (AD). In double mutant amyloid precursor protein/presenilin 2 (APP/PS2) mice, a transgenic model of AD, vessel homeostasis is disturbed. These mice have elevated levels of vascular endothelial growth factor (VEGF) and increased brain endothelial cell division but abnormally low brain vessel density. Examination of the potential involvement of insulin-like growth factor I (IGF-I) in these alterations revealed that treatment with IGF-I, a potent vessel growth promoter in the brain that ameliorates cognitive dysfunction in APP/PS2 mice, counteracted vascular dysfunction as follows: VEGF levels and endothelial cell proliferation were reduced, whereas vascular density was normalized. Notably, abnormally elevated brain IGF-I receptor levels in APP/PS2 mice were also normalized by IGF-I treatment. Analysis of possible processes involved in these alterations indicated that AMP-activated protein kinase (AMPK), a cell energy sensor that intervenes in angiogenic signaling and interacts with IGF-I, was also abnormally activated in APP/PS2 brains. Examination of the consequences of AMPK activation on cultured brain endothelial cells revealed increased VEGF levels together with enhanced endothelial cell proliferation and metabolism. Although these effects were also independently elicited by IGF-I, when both IGF-I and AMPK pathways were simultaneously activated on brain endothelial cells, VEGF production and endothelial cell proliferation ceased while cells remained metabolically activated (glucose use, peroxide production, and mitochondrial activity were elevated) and became more resistant to oxidative stress. Therefore, high IGF-I receptor and phosphoAMPK levels in APP/PS2 brains may reflect imbalanced IGF-I and AMPK angiogenic cross talk that could underlie vascular dysfunction in this model of AD.

ProNGF inhibits NGF-mediated TrkA activation in PC12 cells.

Degeneration of cholinergic basal forebrain neurons (CBFN) is a hallmark in the pathology of Alzheimer's disease (AD). Critically depending upon the neurotrophic support through nerve growth factor (NGF), CBFN in the AD brain face elevated concentrations of the pro-form of NGF (proNGF) and suffer from an imbalance between TrkA and p75(NTR) expression. Research for the underlying mechanisms of CBFN death suggested a pro-apoptotic activity of proNGF. However, this finding could not be confirmed by all investigators and other studies even observed a neurotrophic function of proNGF. In the presence of these controversial findings we investigated the activity of proNGF in PC12 cells with specific emphasis on its neurotoxic versus neurotrophic action. In this study, we show that proNGF can mediate TrkA receptor signaling directly, yet in the manner of a partial agonist with a lower maximum activity than NGF. A pro-apoptotic activity of proNGF could not be confirmed in our cellular system. Interestingly and surprisingly, pre-incubation with proNGF at low, sub-active concentrations inhibited TrkA-mediated neurotrophic NGF signaling in PC12 cells. Our data support a novel hypothesis for the role of elevated proNGF levels in CBFN pathology in AD. Thus, proNGF can indirectly contribute to the slow neurodegeneration in AD by reducing NGF-mediated trophic support.