Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV's putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV−/−) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV+/−) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV−/− and PV+/− mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

Parvalbumin (PV) is a soluble calcium-binding protein that is highly expressed in fast-twitch muscle fibers [1] and specific neurons, including Purkinje cells and GABergic interneurons [2]. Although its putative role acting as a temporary Ca 2+ buffer is still under debate, there is growing evidence that PV is a key player in intracellular Ca 2+ buffering [3,4]. In mammalian fast-twitch muscles, PV facilitates the rapid relaxation by acting as a temporary Ca 2+ buffer [5]. Furthermore, PV-⁄ -fast-twitch muscles were found to be significantly more resistant to fatigue than the wild-type fast-twitch muscles [6]. The fatigue resistance and ability to sustain muscle activity for prolonged periods of time is a principal functional hallmark of slow-twitch type I myofibers (which do not express PV and contain a high fractional volume of mitochondria) because they utilize oxidative metabolism Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a 'slow-onset' Ca 2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV-⁄ -mice. Unexpectedly, PV-⁄ -fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV-⁄ -fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slowtwitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit b revealed the PV-⁄ -tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fasttwitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast-and slow-twitch muscles act as temporary Ca 2+ stores and are thus involved in the shaping of Ca 2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca 2+ transients in PV-⁄ -muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin-and Ca 2+ ⁄ calmodulin-dependent kinase II-dependent pathways. We propose that 'slow-twitch type' mitochondria in PV-⁄ -fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca 2+ removal.

We investigated the role of the two calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) in the locomotor activity and motor coordination using null-mutant mice for PV (PV−/−), CB (CB−/−) or both proteins (PV−/−CB−/−). These proteins are expressed in distinct, mainly non-overlapping populations of neurons of the central and peripheral nervous system and PV additionally in fast-twitch muscles. In a test measuring repeated locomotor activity during 18-20 days, the analysis revealed a slightly increased activity in mice lacking either protein, while the lack of both decreased the number of beams crossed during active periods. An increase in the characteristic speed during the first 8 days could be attributed to PVdeficiency, while the elimination of CB in CB−/− and double-KO mice decreased the percentage of fast movements at all time points. In the latter, additionally a reduction of the fastest speed was observed. The alterations in locomotor activity (fast movements, fastest speed) strongly correlate with the impairment in locomotor coordination in mice deficient for CB evidenced in the runway assay and the rotarod assay. The graded locomotor phenotype (CB > PV) is qualitatively correlated with alterations in Purkinje cell firing reported previously in these mice. The presence or absence of either protein did not affect the spontaneous locomotor activity when animals were placed in a novel environment and tested only once for 30 min. In summary, the lack of these calcium-binding proteins yields characteristic, yet distinct phenotypes with respect to locomotor activity and coordination.

Skeletal muscles show a high plasticity to cope with various physiological demands. Different muscle types can be distinguished by the force, endurance, contraction/relaxation kinetics (fast-twitch vs. slow-twitch muscles), oxidative/glycolytic capacity, and also with respect to Ca2+-signaling components. Changes in Ca2+ signaling and associated Ca2+-dependent processes are thought to underlie the high adaptive capacity of muscle fibers. Here we investigated the consequences and the involved mechanisms caused by the ectopic expression of the Ca2+-binding protein parvalbumin (PV) in C2C12 myotubes in vitro, and conversely, the effects caused by its absence in in fast-twitch muscles of parvalbumin null-mutant (PV−/−) mice in vivo. The absence of PV in fast-twitch muscle tibialis anterior (TA) resulted in an increase in the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and of its positive regulator, the deacetylase sirtuin 1 (SIRT1). TA muscles from PV−/− mice also have an increased mitochondrial volume. Mild ionophore treatment of control (PV-devoid) C2C12 myotubes causing a moderate elevation in [Ca2+]c resulted in an increase in mitochondrial volume, together with elevated PGC-1α and SIRT1 expression levels, whilst it increased PV expression levels in myotubes stably transfected with PV. In PV-expressing myotubes the mitochondrial volume, PGC-1α and SIRT1 were significantly lower than in control C2C12 myotubes already at basal conditions and application of ionophore had no effect on either one. SIRT1 activation causes a down-regulation of PV in transfected myotubes, whilst SIRT1 inhibition has the opposite effect. We conclude that PV expression and mitochondrial volume in muscle cells are inversely regulated via a SIRT1/PGC-1α signaling axis.

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