Magnetogenetic cell activation using endogenous ferritin.

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro . In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo . Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo . Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

Bidirectional Regulation of Motor Circuits Using Magnetogenetic Gene Therapy.

Regulating the activity of discrete neuronal populations in living mammals after delivery of modified ion channels can be used to map functional circuits and potentially treat neurological diseases. Here we report a novel suite of magnetogenetic tools, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity in motor circuits when exposed to magnetic fields. AAV-mediated delivery of a cre-dependent nanobody-TRPV1 calcium channel into the striatum of adenosine 2a (A2a) receptor-cre driver mice led to restricted expression within D2 neurons, resulting in motor freezing when placed in a 3T MRI or adjacent to a transcranial magnetic stimulation (TMS) device. Functional imaging and fiber photometry both confirmed focal activation of the target region in response to the magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing cre into the globus pallidus led to similar circuit specificity and motor responses. Finally, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in subthalamic nucleus (STN) projection neurons in PitX2-cre parkinsonian mice resulted in reduced local c-fos expression and a corresponding improvement in motor rotational behavior during magnetic field exposure. These data demonstrate that AAV delivery of magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits non-invasively in vivo using clinically available devices for both preclinical analysis of circuit effects on behavior and potential human clinical translation.

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells.

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.

Alphaherpesvirus glycoprotein M causes the relocalization of plasma membrane proteins.

Herpesvirus glycoprotein M (gM) is a multiple-spanning integral membrane protein found within the envelope of mature herpesviruses and is conserved throughout the Herpesviridae. gM is defined as a non-essential glycoprotein in alphaherpesviruses and has been proposed as playing a role in controlling final envelopment in a late secretory-pathway compartment such as the trans-Golgi network (TGN). Additionally, gM proteins have been shown to inhibit cell-cell fusion in transfection-based assays by an as yet unclear mechanism. Here, the effect of pseudorabies virus (PRV) gM and the herpes simplex virus type 1 (HSV-1) gM/UL49A complex on the fusion events caused by the HSV-1 glycoproteins gB, gD, gH and gL was investigated. Fusion of cells expressing HSV-1 gB, gD, gH and gL was efficiently inhibited by both PRV gM and HSV-1 gM/UL49A. Furthermore, expression of PRV gM or HSV-1 gM/UL49A, which are themselves localized to the TGN, caused both gD and gH/L to be relocalized from the plasma membrane to a juxtanuclear compartment, suggesting that fusion inhibition is caused by the removal of 'fusion' proteins from the cell surface. The ability of gM to cause the relocalization of plasma membrane proteins was not restricted to HSV-1 glycoproteins, as other viral and non-viral proteins were also affected. These data suggest that herpesvirus gM (gM/N) can alter the membrane trafficking itineraries of a broad range of proteins and this may have multiple functions.