Renzo Mancuso

Amyotrophic lateral sclerosis: Current perspectives from basic research to the clinic

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motoneurons, leading to muscle weakness and paralysis, and finally death. Considerable recent advances have been made in basic research and preclinical therapeutic attempts using experimental models, leading to increasing clinical and translational research in the context of this disease. In this review we aim to summarize the most relevant findings from a variety of aspects about ALS, including evaluation methods, animal models, pathophysiology, and clinical findings, with particular emphasis in understanding the role of every contributing mechanism to the disease for elucidating the causes underlying degeneration of motoneurons and the development of new therapeutic strategies.

Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy

The non-toxic carboxy-terminal fragment of tetanus toxin heavy chain (TTC) has been implicated in the activation of cascades responsible for trophic actions and neuroprotection by inhibition of apoptosis. Previous in vitro studies have described signalling pathways that underlie the administration of TTC to neurons. We investigated whether these properties were maintained in a mouse model of neurodegenerative disease. Naked DNA encoding for TTC was injected intramuscularly and neuromuscular function and clinical behaviour were monitored until endstage in the transgenic SOD1G93A mouse model that expresses a mutant variant of human superoxide dismutase 1 (SOD1). Our results indicate that TTC treatment ameliorated the decline of hindlimb muscle innervation, significantly delayed the onset of symptoms and functional deficits, improved spinal motor neuron survival, and prolonged lifespan. Furthermore, we found that caspase-1 and caspase-3 proapoptotic genes were down-regulated in the spinal cord of treated mice. Western blot analysis showed that the active form of caspase-3 was also down-regulated after TTC treatment and survival signals, such as the significant phosphorylation of serine/threonine protein kinase Akt, were also detected. These results suggest that fragment C of tetanus toxin, TTC, provides a potential therapy for neurodegenerative diseases.

Sigma receptor agonist 2-(4-morpholinethyl)1 phenylcyclohexanecarboxylate (Pre084) increases GDNF and BiP expression and promotes neuroprotection after root avulsion injury

Spinal root avulsion leads to a progressive loss of axotomized motoneurons (MNs). Nowadays, there is no effective treatment to prolong MN survival that could permit recovery as a result of delayed surgical repair. Administration of Sigma-1 receptor (Sig-1R) ligands has been reported to promote beneficial effects after several types of neural injury. In order to shed light of whether Sig-1R ligands could promote MN survival after root avulsion, L4-L5 spinal roots were unilaterally avulsed in adult rats and the Sig-1R agonist Pre084 was administered at different doses. The ventral spinal cords of the animals were studied from 3 to 21 days post-operation (DPO) by using histological, immunohistochemical, and Western blot techniques. Daily treatment with 0.25 mg/kg Pre084 significantly promoted MN survival (68% vs 43% in untreated rats) at 21 DPO, an effect that was antagonized by coadministration of BD1063, an antagonist of Sig-1R. There was a reduction in astroglial- associated immunoreactivity in rats treated with Pre084. Moreover, Pre084 produced an increase in the Sig-1R co-chaperone BiP within MNs, and an increase of GDNF expression by astrocytes in the ventral horn early after injury. Although the mechanisms promoting MN survival by Pre084 remain unclear, we hypothesize that it is mediated at least in part through the increase in these cytoprotective factors. Therefore, early application of Sig-1R agonist appears to be a promising therapy to improve MN survival after root avulsion.

Early presymptomatic cholinergic dysfunction in a murine model of amyotrophic lateral sclerosis

Sporadic and familiar amyotrophic lateral sclerosis (ALS) cases presented lower cholinergic activity than in healthy individuals in their still preserved spinal motoneurons (MNs) suggesting that cholinergic reduction might occur before MN death. To unravel how and when cholinergic function is compromised, we have analyzed the spatiotemporal expression of choline acetyltransferase (ChAT) from early presymptomatic stages of the SOD1G93A ALS mouse model by confocal immunohistochemistry. The analysis showed an early reduction in ChAT content in soma and presynaptic boutons apposed onto MNs (to 76%) as well as in cholinergic interneurons in the lumbar spinal cord of the 30‐day‐old SOD1G93A mice. Cholinergic synaptic stripping occurred simultaneously to the presence of abundant surrounding major histocompatibility complex II (MHC‐II)‐positive microglia and the accumulation of nuclear Tdp‐43 and the appearance of mild oxidative stress within MNs. Besides, there was a loss of neuronal MHC‐I expression, which is necessary for balanced synaptic stripping after axotomy. These events occurred before the selective raise of markers of denervation such as ATF3. By the same time, alterations in postsynaptic cholinergic‐related structures were also revealed with a loss of the presence of sigma‐1 receptor, a Ca2+ buffering chaperone in the postsynaptic cisternae. By 2 months of age, ChAT seemed to accumulate in the soma of MNs, and thus efferences toward Renshaw interneurons were drastically diminished. In conclusion, cholinergic dysfunction in the local circuitry of the spinal cord may be one of the earliest events in ALS etiopathogenesis.

Electrophysiological analysis of a murine model of motoneuron disease.

OBJECTIVE Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motoneurons of the primary motor cortex, the brainstem and the spinal cord, for which there are not effective treatments. Several transgenic mice that mimic motoneuron disease have been used to investigate potential treatments. The objective of this work is to characterize electrophysiologically the SOD1(G93A) transgenic mouse model of ALS, and to provide useful markers to improve early detection and monitoring of progression of the disease. METHODS We performed nerve conduction tests, motor unit number estimation (MUNE), H reflex tests and motor evoked potentials (MEPs) in a cohort of transgenic and wild type mice from 4 to 16 weeks of age. RESULTS The results revealed dysfunction of spinal motoneurons evidenced by deficits in motor nerve conduction tests starting at 8 weeks of age, earlier in proximal than in distal muscles of the hindlimb. MUNE demonstrated that spinal motoneurons loss muscle innervation and have a deficit in their sprouting capacity. Motor evoked potentials revealed that, coexisting with peripheral deficits, there was a dysfunction of central motor tracts that started also at 8 weeks, indicating progressive dysfunction of upper motoneurons. CONCLUSIONS These electrophysiological results provide important information about the SOD1(G93A) mouse model, as they demonstrate by the first time alterations of central motor pathways simultaneously to lower motoneuron dysfunction, well before functional abnormalities appear (by 12 weeks of age). SIGNIFICANCE The finding of concomitant dysfunction of upper and lower motoneurons contributes to the validation of the SOD1(G93A) mouse as model of ALS, because this parallel involvement is a diagnostic condition for ALS. Electrophysiological tests can be used as early markers of the disease and to evaluate the potential benefits of new treatments on both upper and lower motoneurons.

Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule.

BackgroundAmyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases. Neurotrophic factors have been widely tested to counteract neurodegenerative conditions, despite their unspecific neuronal access. The non-toxic C-terminal fragment of the tetanus toxin (TTC) heavy chain has been studied not only as a carrier molecule to the CNS but also as a neuroprotective agent. Because the neurotrophic effects of BDNF have been demonstrated in vitro and in vivo, the question addressed in this work is whether a fusion molecule of BDNF-TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone in a mouse model of ALS.MethodsRecombinant plasmid constructs (pCMV-TTC and pCMV-BDNF-TTC) were injected into the quadriceps femoris and triceps brachialis muscles of SOD1G93A transgenic mice at 8 weeks of age. The hanging wire and rotarod tests were performed to assess motor coordination, strength and balance. Electrophysiological tests, morphological assays of spinal cord sections of L2 and L4 segments, and gene and protein expression analyses were performed. The Kaplan-Meier survival analysis test was used for comparisons of survival. Multiple comparisons of data were analyzed using a one-way analysis of variance (ANOVA).ResultsTreatment with the fusion-molecule BDNF-TTC and with TTC alone significantly delayed the onset of symptoms and functional deficits of SOD1G93A mice. Muscle innervation was partially preserved with these treatments, and the number of surviving motoneurons in L2 spinal cord segment was increased particularly by the fusion protein induction. Inhibition of pro-apoptotic protein targets (caspase-3 and Bax) and significant phosphorylation of Akt and ERK were also found in the spinal cord of treated mice.ConclusionsSignificant improvements in behavioral and electrophysiological results, motoneuron survival and anti-apoptotic/survival-activated pathways were observed with BDNF-TTC treatment. However, no synergistic effect was found for this fusion molecule. Although BDNF in the fusion molecule is capable of activating autocrine and neuroprotective pathways, TTC treatment alone yielded similar neuroprotection. Therefore, an accurate study of the neuroprotective effects of TTC fusion molecules should be performed to obtain a better understanding of its effects.

CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves

Inflammation is a common neuropathological feature in several neurological disorders, including amyotrophic lateral sclerosis (ALS). We have studied the contribution of CSF1R signalling to inflammation in ALS, as a pathway previously reported to control the expansion and activation of microglial cells. We found that microglial cell proliferation in the spinal cord of SOD1G93A transgenic mice correlates with the expression of CSF1R and its ligand CSF1. Administration of GW2580, a selective CSF1R inhibitor, reduced microglial cell proliferation in SOD1G93A mice, indicating the importance of CSF1-CSF1R signalling in microgliosis in ALS. Moreover, GW2580 treatment slowed disease progression, attenuated motoneuron cell death and extended survival of SOD1G93A mice. Electrophysiological assessment revealed that GW2580 treatment protected skeletal muscle from denervation prior to its effects on microglial cells. We found that macrophages invaded the peripheral nerve of ALS mice before CSF1R-induced microgliosis occurred. Interestingly, treatment with GW2580 attenuated the influx of macrophages into the nerve, which was partly caused by the monocytopenia induced by CSF1R inhibition. Overall, our findings provide evidence that CSF1R signalling regulates inflammation in the central and peripheral nervous system in ALS, supporting therapeutic targeting of CSF1R in this disease.

Randall-Selitto Test: A New Approach for the Detection of Neuropathic Pain after Spinal Cord Injury

In this work we assess the usefulness of the Randall-Selitto test as a method to detect and quantify neuropathic pain responses in rats subjected to different spinal cord injuries. The mechanical nociceptive thresholds were significantly reduced during follow-up after spinal cord contusion or transection. Our results demonstrate that the Randall-Selitto test allows the detection of neuropathic pain both in forepaws and hindpaws, as well as in dorsal and plantar surfaces. Moreover, it does not require weight support capacity, so it can be used at early time points after the injury. This is the first time that this method has been used to describe the changes in nociceptive thresholds that take place after spinal cord injuries of different severities over time.

Effect of genetic background on onset and disease progression in the SOD1-G93A model of amyotrophic lateral sclerosis

Abstract Knowledge of the potential effect of genetic background in disease models is important. The SOD1-G93A transgenic mouse is the most widely used model in amyotrophic lateral sclerosis (ALS). Since these animals show considerable variability both in the onset and the progression of the disease, this study aimed to characterize the potential differences between the two most widely used strains, C56BL/6 (B6) and B6SJL. A rotarod test was carried out to assess strength and motor coordination, while electrophysiology tests were performed to evaluate the function of upper and lower motor neurons. Survival of the animals and motor neuron loss were also studied. The results did not show any background effect regarding the rotarod test, despite the differences in the pattern of decline in central and peripheral motor conduction. The onset of motor neuron abnormalities was later in B6SJL mice, but progressed more rapidly. Lifespan was longer for B6 than for B6SJL animals. In conclusion, background differences in disease onset and progression are important. The characteristics of the strain should be taken into account in experimental design of therapeutic studies.

Differential effects on KCC2 expression and spasticity of ALS and traumatic injuries to motoneurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease manifested by progressive muscle atrophy and paralysis due to the loss of upper and lower motoneurons (MN). Spasticity appears in ALS patients leading to further disabling consequences. Loss of the inhibitory tone induced by downregulation of the potassium chloride cotransporter 2 (KCC2) in MN has been proposed to importantly contribute to the spastic behavior after spinal cord injury (SCI). The aim of the present study was to test whether the alterations in the expression of KCC2 are linked to the appearance of spasticity in the SODG93A ALS murine model. We compared SODG93A mice to wild type mice subjected to SCI to mimic the spinal MN disconnection from motor descending pathways, and to sciatic nerve lesion to mimic the loss of MN connectivity to muscle. Electrophysiological results show that loss of motor function is observed at presymptomatic stage (8 weeks) in SODG93A mice but hyperreflexia and spasticity do not appear until a late stage (16 weeks). However, KCC2 was not downregulated despite MN suffered disconnection both from muscles and upper MNs. Further experiments revealed decreased gephyrin expression, as a general marker of inhibitory systems, accompanied by a reduction in the number of Renshaw interneurons. Moreover, 5-HT fibers were increased in the ventral horn of the lumbar spinal cord at late stage of disease progression in SOD1G93A mice. Taken together, the present results indicate that spasticity appears late in the ALS model, and may be mediated by a decrease in inhibitory interneurons and an increase of 5-HT transmission, while the absence of down-regulation of KCC2 could rather indicate an inability of MNs to respond to insults.