Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile-profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH-like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH-like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations

Parvalbumin and somatostatin interneurons contribute to the generation of hippocampal gamma oscillations

γ-frequency oscillations (30-120 Hz) in cortical networks influence neuronal encoding and information transfer, and are disrupted in multiple brain disorders. While synaptic inhibition is important for synchronization across the γ-frequency range, the role of distinct interneuronal subtypes in slow (<60 Hz) and fast γ states remains unclear. Here, we used optogenetics to examine the involvement of parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons in γ oscillations in the mouse hippocampal CA3 ex vivo, using animals of either sex. Disrupting either PV+ or SST+ interneuron activity, via either photoinhibition or photoexcitation, led to a decrease in the power of cholinergically induced slow γ oscillations. Furthermore, photoexcitation of SST+ interneurons induced fast γ oscillations, which depended on both synaptic excitation and inhibition. Our findings support a critical role for both PV+ and SST+ interneurons in slow hippocampal γ oscillations, and further suggest that intense activation of SST+ interneurons can enable the CA3 circuit to generate fast γ oscillations

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons

Parvalbumin (PV+) interneurons constitute a small proportion of the total neuronal population (less than 2% in the hippocampus), yet they possess crucial roles in shaping neuronal network activity (Freund and Buzsáki, 1996; Jonas et al., 2004; Pelkey et al., 2017). PV+ interneurons inhibit their postsynaptic targets efficiently by applying fast perisomatic inhibition and have been directly implicated in the generation of network activity at the gamma (γ) band frequency (30–80 Hz) (Antonoudiou et al., 2020; Cardin et al., 2009; Hájos et al., 2004; Mann et al., 2005; Sohal et al., 2009). Network oscillations at γ-band frequency are believed to facilitate information transmission through circuit synchronization and local gain control that may be instrumental in multiple cognitive processes such as attention, learning, and memory (Akam and Kullmann, 2010; Fries, 2015; Howard et al., 2003; Montgomery and Buzsaki, 2007; Sohal, 2016). Importantly, these oscillations are thought to be metabolically very costly, and it has therefore been postulated that PV+ interneurons require substantial amounts of energy via ATP hydrolysis to sustain the high firing rate and dissipate ion gradients during neuronal transmission (Attwell and Laughlin, 2001; Kann, 2011; Kann, 2016; Kann and Kovács, 2007; Kann et al., 2014). Thus, it is crucial to understand the metabolic expenditure and the involvement of mitochondria in PV+ interneurons. Indeed, electron microscopy, histochemical, and transcriptomic approaches have revealed that PV+ interneurons have a higher density of energy-producing mitochondria and elevated expression levels of electron transport chain components (Adams et al., 2015; Gulyás et al., 2006; Nie and Wong-Riley, 1995; Paul et al., 2017). The spatiotemporal organization of mitochondria is essential for the precise provision of ATP and Ca2+-buffering for neuronal transmission and communication (Devine and Kittler, 2018; MacAskill and Kittler, 2010). Miro1 is a mitochondrial adaptor protein, responsible for coupling mitochondria to the cytoskeleton and for their bidirectional trafficking in axons and dendrites (Birsa et al., 2013; Guo et al., 2005; López-Doménech et al., 2016; López-Doménech et al., 2018; Macaskill et al., 2009; Nguyen et al., 2014; Saotome et al., 2008; Wang and Schwarz, 2009). Global deletion of Miro1 (encoded by the Rhot1 gene) is perinatal lethal, while the conditional removal of Miro1 from cortical and hippocampal pyramidal cells alters the occupancy of dendritic mitochondria due to impairment in trafficking, resulting in dendritic degeneration and cell death (López-Doménech et al., 2016). In contrast, the significance of mitochondrial trafficking and distribution in PV+ interneurons, and the role of Miro1, is completely unexplored and especially interesting as their axon is highly branched with a cumulative length reaching up to 50 mm in the hippocampus (Hu et al., 2014). In this study, we generated a transgenic mouse line where mitochondria are fluorescently labelled in PV+ interneurons. We crossed this line with the Miro1 floxed mouse (Rhot1flox/flox), to generate a model where Miro1 was conditionally knocked-out exclusively in PV+ interneurons. Using two-photon live-imaging of ex vivo organotypic brain slices, we demonstrated a reduction in mitochondrial trafficking in the absence of Miro1 in PV+ interneurons in the hippocampus. The impairment in Miro1-directed mitochondrial transport led to an accumulation of mitochondria in the soma and their depletion from axonal presynaptic terminals in acute hippocampal brain slices. Loss of Miro1 resulted in alterations in axonal but not dendritic branching in PV+ interneurons. While the ability of PV+ interneurons to apply long-lasting inhibition to post-synaptic targets remained intact, the changes in Miro1-dependent mitochondrial dynamics were accompanied by an increased frequency of γ-oscillations in hippocampal brain slices and a reduction in anxiety-related emotional behavior. Thus, we show that Miro1-dependent mitochondrial positioning is essential for correct PV+ interneuron function, network activity, and anxiolytic animal behavior