Chiara Rossi

Long-Distance Retrograde Effects of Botulinum Neurotoxin A

Botulinum neurotoxins (designated BoNT/A–BoNT/G) are bacterial enzymes that block neurotransmitter release by cleaving essential components of the vesicle fusion machinery. BoNT/A, which cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), is extensively exploited in clinical medicine to treat neuromuscular pathologies, facial wrinkles, and various types of pain. It is widely assumed that BoNT/A remains at the synaptic terminal and its effects are confined to the injection site. Here we demonstrate that catalytically active BoNT/A is retrogradely transported by central neurons and motoneurons and is then transcytosed to afferent synapses, in which it cleaves SNAP-25. SNAP-25 cleavage by BoNT/A was observed in the contralateral hemisphere after unilateral BoNT/A delivery to the hippocampus. Appearance of cleaved SNAP-25 resulted in blockade of hippocampal activity in the untreated hemisphere. Injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the retina. Cleaved SNAP-25 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles. Experiments excluded passive spread of the toxin and demonstrated axonal migration and neuronal transcytosis of BoNT/A. These findings reveal a novel pathway of BoNT/A trafficking in neurons and have important implications for the clinical uses of this neurotoxin.

Remodeling of second-order neurons in the retina of rd/rd mutant mice

This is a brief review of data obtained by analyzing the morphology and the physiology of the retinas in rd/rd and normal, wt mice, aged 10-90 days. Second-order neurons of the rd/rd show abnormalities that start with the anomalous development of rod bipolar cells around P10 and culminate with the atrophy of dendrites in cone bipolar cells, mostly evident at P90. Horizontal cells remodel considerably. Cone-mediated ERGs, (recorded between 13 and 16 days of age) have reduced a-wave and b-wave amplitudes and longer b-wave latency and duration. B-wave abnormalities indicate specific postreceptoral dysfunction. Morphological and ERG changes in rd/rd retinas are consistent with substantial inner retinal remodeling associated to photoreceptor degeneration.

Evidence for Anterograde Transport and Transcytosis of Botulinum Neurotoxin A (BoNT/A)

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that blocks synaptic transmission via the cleavage of SNAP-25 (synaptosomal-associated protein of 25 kDa). BoNT/A is successfully used in clinical neurology for the treatment of several neuromuscular pathologies and pain syndromes. Despite its widespread use, relatively little is known on BoNT/A intracellular trafficking in neurons. Using the visual pathway as a model system, here we show that catalytically active BoNT/A is capable of undergoing anterograde axonal transport and transcytosis. Following BoNT/A injection into the rat eye, significant levels of BoNT/A-cleaved SNAP-25 appeared in the retinorecipient layers of the superior colliculus (SC). Anterograde propagation of BoNT/A effects required axonal transport, ruling out a systemic spread of the toxin. Cleaved SNAP-25 was present in presynaptic structures of the tectum, but retinal terminals were devoid of the immunoreactivity, indicative of transcytosis. Experiments based on sequential administration of BoNT/A and BoNT/E showed a persistent catalytic activity of BoNT/A in tectal cells following its injection into the retina. Our findings demonstrate that catalytically active BoNT/A is anterogradely transported from the eye to the SC and transcytosed to tectal synapses. These data are important for a more complete understanding of the mechanisms of action of BoNT/A.

Transient Synaptic Silencing of Developing Striate Cortex Has Persistent Effects on Visual Function and Plasticity

Neural circuits in the cerebral cortex are shaped by experience during “critical periods” early in life. For example, visual cortex is immature at the time of eye opening and gradually develops its functional properties during a sensitive period. Very few reports have addressed the role of intrinsic neural activity in cortical maturation. Here we have exploited the bacterial enzyme botulinum neurotoxin E (BoNT/E) to produce a unilateral, reversible blockade of neural activity in rat visual cortex during the sensitive period. BoNT/E is a highly selective protease that interferes with transmitter release via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Unilateral, intracortical injections of BoNT/E were made at the time of eye opening and resulted in the silencing of the treated, but not contralateral, hemisphere for a period of 2 weeks. We found that visual acuity was permanently reduced in the blocked hemisphere, and the critical period for ocular dominance plasticity persisted into adulthood. Unexpectedly, these effects extended equally to the contralateral, uninjected side, demonstrating a fundamental role for interhemispheric connections in cortical maturation.

Macro-EMG and MUNE Changes in Patients with Amyotrophic Lateral Sclerosis: One-Year Follow Up

ABSTRACT Objective: We assessed changes in Motor Units (MU) and extent of MU loss using macro-electromyography (macro-EMG) and Motor Unit Number Estimation (MUNE). Methods: We applied these techniques to a sample of 61 Amyotrophic Lateral Sclerosis (ALS) patients basally (T0) and after 4 (T1), 8 (T2), and 12 (T3) months. Macro Motor Unit Potentials (macro-MUPs) were derived from Biceps Brachii (BB) muscle; MUNE was performed both in BB and Abductor Digiti Minimi (ADM) muscles of the same side. Results: Macro-MUPs area resulted in progressive increase at T1, T2, and T3 with respect to T0. Fiber density (FD) at T3 decreases a bit than at T2. Functioning MUS number decreased in both the muscles throughout the entire follow-up with respect to T0 and the rate of MU decrease was similar in both the muscles, but steeper distally. Conclusions and Significance: Macro-EMG increase and FD decrease suggest that a process of MU rearrangement begins to fall after 8 months of disease course. Combined use of macro-EMG and MUNE techniques in ALS patients allows to track over time changes in muscle MU features and number in face of progressive anterior horn cells death during diseases evolution.

Environmental enrichment promotes fiber sprouting after deafferentation of the superior colliculus in the adult rat brain.

Exposure to an enriched environment has proven to be beneficial in the recovery of function after brain lesions, but the underlying mechanisms remain only partly understood. One possibility is that environmental enrichment stimulates the reorganization of areas and fiber tracts that have been spared by the injury. Here we evaluate the effects of enriched environment on the sprouting of undamaged retinal afferents into the deafferented superior colliculus (SC) after a partial retinal lesion in adult rats. Anterograde tracing of retinal axons demonstrated a significant increase in fiber sprouting in the denervated SC of animals reared in enriched environment compared to animals reared in standard conditions. Environmental enrichment also promoted a substantial recovery of synaptic sites within the deafferented SC as shown by both synapsin I and vesicular glutamate transporter 2 immunostaining. These data provide evidence that environmental enrichment stimulates axonal plasticity and synaptic reorganization following brain injury.

How to Assess Disease’s Severity and Monitor Patients with Amyotrophic Lateral Sclerosis: Lessons from Neurophysiology

Ferdinando Sartucci1,2,3, Tommaso Bocci1,4, Lucia Briscese1, Chiara Pecori1,3, Chiara Rossi1 and Fabio Giannini4 1Department of Neuroscience, Unit of Neurology, Pisa University Medical School, 2Institute of Neuroscience, CNR, Pisa, 3Department of Neuroscience, Unit Outpatients Neurological Activity, Pisa University Medical School, Pisa, 4Department of Neurological Neurosurgical and Behavioural Sciences, Siena University Medical School, Siena, Italy