Nerve pathology is prevented by linker proteins in mouse models for LAMA2-related muscular dystrophy.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is a devastating congenital muscular dystrophy that is caused by mutations in the LAMA2 gene encoding laminin-α2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by an early-onset, severe muscular dystrophy that ultimately leads to death by respiratory insufficiency. However, pathology in nonmuscle tissues has been described. Prior work in the dyW /dyW mouse model for LAMA2 MD has shown that two linker proteins, mini-agrin and αLNNd, when expressed in skeletal muscle fibers, greatly increase survival from a few months up to more than 2 years. However, the restoration of skeletal muscle function accentuates the pathology in nonmuscle tissue in dyW /dyW mice, first and foremost in the peripheral nerve resulting in paralysis of the hind limbs. We now show that the expression of the two linker proteins in all tissues ameliorates the muscular dystrophy and prevents the appearance of the hind limb paralysis. Importantly, the same ameliorating effect of the linker proteins was seen in dy3K /dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-α2 in muscle and peripheral nerve, which are the two organs most affected in LAMA2 MD. These results are of key importance for designing appropriate expression constructs for mini-agrin and αLNNd to develop a gene therapy for LAMA2 MD patients.

JAB1 deletion in oligodendrocytes causes senescence-induced inflammation and neurodegeneration in mice.

Oligodendrocytes are the primary target of demyelinating disorders, and progressive neurodegenerative changes may evolve in the CNS. DNA damage and oxidative stress are considered key pathogenic events, but the underlying molecular mechanisms remain unclear. Moreover, animal models do not fully recapitulate human diseases, complicating the path to effective treatments. Here we report that mice with cell-autonomous deletion of the nuclear COP9 signalosome component CSN5 (JAB1) in oligodendrocytes develop DNA damage and defective DNA repair in myelinating glial cells. Interestingly, oligodendrocytes lacking JAB1 expression underwent a senescence-like phenotype that fostered chronic inflammation and oxidative stress. These mutants developed progressive CNS demyelination, microglia inflammation, and neurodegeneration, with severe motor deficits and premature death. Notably, blocking microglia inflammation did not prevent neurodegeneration, whereas the deletion of p21CIP1 but not p16INK4a pathway ameliorated the disease. We suggest that senescence is key to sustaining neurodegeneration in demyelinating disorders and may be considered a potential therapeutic target.

Mesoangioblast delivery of miniagrin ameliorates murine model of merosin-deficient congenital muscular dystrophy type 1A.

BACKGROUND: Merosin-deficient congenital muscular dystrophy type-1A (MDC1A) is characterized by progressive muscular dystrophy and dysmyelinating neuropathy caused by mutations of the α2 chain of laminin-211, the predominant laminin isoform of muscles and nerves. MDC1A has no available treatment so far, although preclinical studies showed amelioration of the disease by the overexpression of miniagrin (MAG). MAG reconnects orphan laminin-211 receptors to other laminin isoforms available in the extracellular matrix of MDC1A mice. METHODS: Mesoangioblasts (MABs) are vessel-associated progenitors that can form the skeletal muscle and have been shown to restore defective protein levels and motor skills in animal models of muscular dystrophies. As gene therapy in humans still presents challenging technical issues and limitations, we engineered MABs to overexpress MAG to treat MDC1A mouse models, thus combining cell to gene therapy. RESULTS: MABs synthesize and secrete only negligible amount of laminin-211 either in vitro or in vivo. MABs engineered to deliver MAG and injected in muscles of MDC1A mice showed amelioration of muscle histology, increased expression of laminin receptors in muscle, and attenuated deterioration of motor performances. MABs did not enter the peripheral nerves, thus did not affect the associated peripheral neuropathy. CONCLUSIONS: Our study demonstrates the potential efficacy of combining cell with gene therapy to treat MDC1A.

Jab1 regulates Schwann cell proliferation and axonal sorting through p27.

Axonal sorting is a crucial event in nerve formation and requires proper Schwann cell proliferation, differentiation, and contact with axons. Any defect in axonal sorting results in dysmyelinating peripheral neuropathies. Evidence from mouse models shows that axonal sorting is regulated by laminin211- and, possibly, neuregulin 1 (Nrg1)-derived signals. However, how these signals are integrated in Schwann cells is largely unknown. We now report that the nuclear Jun activation domain-binding protein 1 (Jab1) may transduce laminin211 signals to regulate Schwann cell number and differentiation during axonal sorting. Mice with inactivation of Jab1 in Schwann cells develop a dysmyelinating neuropathy with axonal sorting defects. Loss of Jab1 increases p27 levels in Schwann cells, which causes defective cell cycle progression and aberrant differentiation. Genetic down-regulation of p27 levels in Jab1-null mice restores Schwann cell number, differentiation, and axonal sorting and rescues the dysmyelinating neuropathy. Thus, Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation. Finally, Jab1 may constitute a key molecule in the pathogenesis of dysmyelinating neuropathies.

Vimentin regulates peripheral nerve myelination.

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.

Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies.

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

Over-expression of amyloid precursor protein in HEK cells alters p53 conformational state and protects against doxorubicin.

Here we show that human embryonic kidney (HEK) cells stably transfected with amyloid precursor protein (HEK-APP), expressed a conformational mutant-like and transcriptionally inactive p53 isoform, and turned out to be less sensitive to the cytotoxin doxorubicin in comparison with untransfected cells. Treatment of HEK-APP cells with gamma- and beta-secretase inhibitors prevented generation of unfolded, mutant-like p53 isoform and made the cells vulnerable to doxorubicin as untransfected cells. Changes in p53 conformational state and reduced sensitivity to doxorubicin were also found in untransfected HEK cells after exposure to nanomolar concentrations of beta-amyloid (Abeta) and these effects were antagonized by vitamin E. The modulator effects of Abeta on p53 conformational state were, at least in part, due to the intracellular peptides as (i) treatment of HEK-APP cells with an antibody that sequestered extracellular Abeta did not modify the capability of the cells to express the mutant-like p53 isoform; (ii) in the presence of 1% serum exogenous Abeta peptide crossed the plasma membrane, as demonstrated by confocal analysis and ELISA, and induced p53 conformational change; and (iii) in the presence of 10% serum Abeta did not enter the cells and consequently did not influence the p53 conformational state.

Role of acetylcholinesterase inhibitors in pharmacological regulation of amyloid precursor protein processing.

The triggering events leading to the selective neurodegeneration observed in Alzheimer brains are not yet completely understood. They thus create a great challenge for the definition of a resolutive treatment for the causes and symptoms of Alzheimer's Disease (AD). Since the current therapeutic option for AD patients is the use of acetylcholinesterase inhibitors (AChEIs), several authors have examined whether these drugs can also affect the expression and metabolism of the amyloid precursor protein (AbetaPP). The rationale behind these studies was based on the fact that the literature suggests that cholinergic activities are also involved in the regulation of AbetaPP metabolism. Therefore, the characterization of these aspects of AD pharmacology may allow cholinergic drugs to be tested for their ability to intervene at different levels of the pathogenetic chain, other than providing a replacement therapy for lost neurotransmitters. This paper reviews the evidence that many of these drugs, although with different qualitative effects, are able to modulate the metabolism and expression of AbetaPP. This effect is often sustained by an indirect cholinergic mechanism and does not affect the mRNA expression of the precursor, although some other authors have demonstrated an effect on post-transcriptional regulation of AbetaPP expression. In addition to the effect on AbetaPP processing, we recently explored the possibility that these molecules affect a gene expression program beyond the classical pharmacological effects, for insights on possibly unexplored pathways of intervention in AD.

Role of acetylcholinesterase inhibitors in the regulation of amyloid beta precursor protein (AbetaPP) metabolism.

The events that lead to neurodegeneration in Alzheimer's brains are largely unknown and this fact creates a great challenge for the definition of treatments that may be resolutive for Alzheimer's disease (AD). The current therapeutic option for AD patients is the use of acetylcholinesterase inhibitors (AChEIs), which gives a symptomatic relief to some of the clinical manifestations of the disease. In addition, several authors investigated whether these drugs can also affect one of the major pathogenetic pathway postulated for the disease, that is the expression and metabolism of the amyloid precursor protein (AbetaPP). The literature suggests that cholinergic activities may be significantly involved in the regulation of AbetaPP metabolism, thus the characterisation of these aspects of AD pharmacology may allow testing cholinergic drugs in their ability to intervene at different levels of the pathogenetic chain, other than to provide a replacement therapy for lost neurotransmitters. In this paper we review the evidence that these drugs, albeit with different quantitative and qualitative effects, can modulate the metabolism and expression of AbetaPP, through mechanism that involve either an indirect cholinergic mechanism or effects that do not involve the canonical pharmacological activity of AChE inhibitors. We also provide preliminary evidence that these molecules may affect a gene expression program beyond the classical pharmacological pattern suggesting an insight on possible unexplored pathways of intervention in AD.

Acetylcholinesterase inhibitors: novel activities of old molecules.

The therapeutic approach for improving the cognitive function in patients with Alzheimer's disease (AD) is mainly based on the potentiation of central cholinergic activity and is achieved clinically by the use of acetylcholinesterase (AChE) inhibitors such as tacrine, donepezil, rivastigmine, galantamine and other drugs currently in clinical trials. These are, by their pharmacology, only symptomatic drugs yet recently these molecules have shown some potential also in the modulation of amyloid precursor protein (APP) processing. We explore in this review the experimental evidence that suggests a role for AChEIs in APP processing and point to multiple complex mechanisms involving either a cholinergic agonist effect, coupled to multiple signal transduction pathways, or post-transcriptional effects that modulate the expression of cellular APP.

Differential involvement of protein kinase C alpha and epsilon in the regulated secretion of soluble amyloid precursor protein.

We investigated the differential role of protein kinase C (PKC) isoforms in the regulated proteolytic release of soluble amyloid precursor protein (sAPPalpha) in SH-SY5Y neuroblastoma cells. We used cells stably transfected with cDNAs encoding either PKCalpha or PKCepsilon in the antisense orientation, producing a reduction of the expression of PKCalpha and PKCepsilon, respectively. Reduced expression of PKCalpha and/or PKCepsilon did not modify the response of the kinase to phorbol ester stimulation, demonstrating translocation of the respective isoforms from the cytosolic fraction to specific intracellular compartments with an interesting differential localization of PKCalpha to the plasma membrane and PKCepsilon to Golgi-like structures. Reduced expression of PKCalpha significantly impaired the secretion of sAPPalpha induced by treatment with phorbol esters. Treatment of PKCalpha-deficient cells with carbachol induced a significant release of sAPPalpha. These results suggest that the involvement of PKCalpha in carbachol-induced sAPPalpha release is negligible. The response to carbachol is instead completely blocked in PKCepsilon-deficient cells suggesting the importance of PKCepsilon in coupling cholinergic receptors with APP metabolism.

Familial migraine with aura: association study with 5-HT1B/1D, 5-HT2C, and hSERT polymorphisms.

BACKGROUND: The serotonergic system has a significant role in the pathophysiology and pharmacology of migraine. OBJECTIVE: To study the association between the occurrence of migraine with aura and 5-HT(1B/1D) and 5-HT(2C) receptor gene and the human serotonin transporter (hSERT) gene polymorphisms in 18 unrelated families with multiple affected members. METHOD: Two polymorphisms in the 5-HT(1B/1D) receptor gene and one polymorphism in the 5-HT(2C) receptor gene were studied by restriction fragment length polymorphism analysis. Allelic variation of the hSERT, with 9, 10, and 12 copies of a "repetitive element," was studied by polymerase chain reaction amplification of the variable number tandem repeat region. RESULTS: Allelic distribution of 5-HT(1B/1D) and 5-HT(2C) receptor gene polymorphisms in affected patients did not differ in either of the control groups (unaffected relatives or unrelated healthy individuals). A trend toward a significant effect of the 12-repeat hSERT allele as a risk factor for migraine with aura versus unrelated controls was observed. CONCLUSION: Our data do not support the involvement of 5-HT(1B/1D) and 5-HT(2C) receptor gene polymorphisms in migraine with aura, yet do suggest a possible role for a locus at or near the hSERT gene in the susceptibility to migraine with aura.