Caffeine alleviates progressive motor deficits in a transgenic mouse model of spinocerebellar ataxia.

OBJECTIVE: Machado-Joseph disease (MJD) is a neurodegenerative spinocerebellar ataxia (SCA) associated with an expanded polyglutamine tract within ataxin-3 for which there is currently no available therapy. We previously showed that caffeine, a nonselective adenosine receptor antagonist, delays the appearance of striatal damage resulting from expression of full-length mutant ataxin-3. Here we investigated the ability of caffeine to alleviate behavioral deficits and cerebellar neuropathology in transgenic mice with a severe ataxia resulting from expression of a truncated fragment of polyglutamine-expanded ataxin-3 in Purkinje cells. METHODS: Control and transgenic c57Bl6 mice expressing in the mouse cerebella a truncated form of human ataxin-3 with 69 glutamine repeats were allowed to freely drink water or caffeinated water (1g/L). Treatments began at 7 weeks of age, when motor and ataxic phenotype emerges in MJD mice, and lasted up to 20 weeks. Mice were tested in a panel of locomotor behavioral paradigms, namely rotarod, beam balance and walking, pole, and water maze cued-platform version tests, and then sacrificed for cerebellar histology. RESULTS: Caffeine consumption attenuated the progressive loss of general and fine-tuned motor function, balance, and grip strength, in parallel with preservation of cerebellar morphology through decreasing the loss of Purkinje neurons and the thinning of the molecular layer in different folia. Caffeine also rescued the putative striatal-dependent executive and cognitive deficiencies in MJD mice. INTERPRETATION: Our findings provide the first in vivo demonstration that caffeine intake alleviates behavioral disabilities in a severely impaired animal model of SCA. Ann Neurol 2017;81:407-418.

Caffeine and adenosine A(2A) receptor inactivation decrease striatal neuropathology in a lentiviral-based model of Machado-Joseph disease.

OBJECTIVE: Machado-Joseph disease (MJD) is a neurodegenerative disorder associated with an abnormal CAG expansion, which translates into an expanded polyglutamine tract within ataxin-3. There is no therapy to prevent or modify disease progression. Because caffeine (a nonselective adenosine receptor antagonist) and selective adenosine A2A receptor (A2A R) blockade alleviate neurodegeneration in different brain diseases, namely at early stages of another polyglutamine-related disorder such as Huntington's disease, we now tested their ability to control MJD-associated neurodegeneration. METHODS: MJD was modeled by transducing the striatum of male adult C57Bl/6 mice with lentiviral vectors encoding mutant ataxin-3 in one hemisphere and wild-type ataxin-3 in the other hemisphere (as internal control). Caffeine (1g/L) was applied through the drinking water. Mice were killed at different time points (from 2 to 12 weeks) to probe for the appearance of different morphological changes using immunohistochemical analysis. RESULTS: Mutant ataxin-3 caused an evolving neuronal dysfunction (loss of DARPP-32 staining) leading to neurodegeneration (cresyl violet and neuronal nuclei staining) associated with increased number of mutant ataxin-3 inclusions in the basal ganglia. Notably, mutant ataxin-3 triggered early synaptotoxicity (decreased synaptophysin and microtubule-associated protein-2 staining) and reactive gliosis (glial fibrillary acidic protein and CD11b staining), which predated neuronal dysfunction and damage. Caffeine reduced the appearance of all these morphological modifications, which were also abrogated in mice with a global A2A R inactivation (knockout). INTERPRETATION: Our findings provide a demonstration that synaptotoxicity and gliosis are precocious events in MJD and that caffeine and A2A R inactivation decrease MJD-associated striatal pathology, which paves the way to consider A2A Rs as novel therapeutic targets to manage MJD.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Adenosine A2A Receptors in the Amygdala Control Synaptic Plasticity and Contextual Fear Memory.

The consumption of caffeine modulates working and reference memory through the antagonism of adenosine A2A receptors (A2ARs) controlling synaptic plasticity processes in hippocampal excitatory synapses. Fear memory essentially involves plastic changes in amygdala circuits. However, it is unknown if A2ARs in the amygdala regulate synaptic plasticity and fear memory. We report that A2ARs in the amygdala are enriched in synapses and located to glutamatergic synapses, where they selectively control synaptic plasticity rather than synaptic transmission at a major afferent pathway to the amygdala. Notably, the downregulation of A2ARs selectively in the basolateral complex of the amygdala, using a lentivirus with a silencing shRNA (small hairpin RNA targeting A2AR (shA2AR)), impaired fear acquisition as well as Pavlovian fear retrieval. This is probably associated with the upregulation and gain of function of A2ARs in the amygdala after fear acquisition. The importance of A2ARs to control fear memory was further confirmed by the ability of SCH58261 (0.1 mg/kg; A2AR antagonist), caffeine (5 mg/kg), but not DPCPX (0.5 mg/kg; A1R antagonist), treatment for 7 days before fear conditioning onwards, to attenuate the retrieval of context fear after 24-48 h and after 7-8 days. These results demonstrate that amygdala A2ARs control fear memory and the underlying process of synaptic plasticity in this brain region. This provides a neurophysiological basis for the association between A2AR polymorphisms and phobia or panic attacks in humans and prompts a therapeutic interest in A2ARs to manage fear-related pathologies.