The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calfserum for 2-12 days in vtitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and ofnewly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K+ concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K+ concentration was small and Ca 2 independent, but it progressively and steadily increased (up to 8-to 10-fold) and became Ca2" dependent (up to 80-85%) at later stages (4, 8, and 12 DIV).Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. y-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that ofglutamate, and y-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K+ concentration-evoked release of this amino acid was modest and scarcely affected by Ca2". Neither high K+ concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.It is known that the excitatory information reaching the cerebellum through the mossy fibers is conveyed to the inhibitory neurons of the cerebellar cortex (mainly to the Purkinje cells) by excitatory interneurones-the granule cells (1), which represent the most abundant neuronal population of the cerebellar cortex. Although glutamic acid has been proposed as the excitatory transmitter of granule cells (2-7), largely on the basis of indirect neurochemical evidence, definitive proof substantiating this hypothesis is still lacking, and some data ofthe literature even appear to be in contrast with it. For example, the high affinity uptake and the concentration of glutamate were not found to be significantly decreased in cerebella severely depleted of granule cells (refs. 5 and 8; however, see refs. 2-7), as one would expect if a substantial glutamate pool were associated with these cells. Moreover, autoradiographic studies on cerebellar slices preincubated in the presence of low concentrati...

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Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and "neuron-like" y-aminobutyric acid transport (glial

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimer's disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.