Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Sex differences in mouse infralimbic cortex projections to the nucleus accumbens shell.

BACKGROUND: The nucleus accumbens (NAc) is an important region in motivation and reward. Glutamatergic inputs from the infralimbic cortex (ILC) to the shell region of the NAc (NAcSh) have been implicated in driving the motivation to seek reward through repeated action-based behavior. While this has primarily been studied in males, observed sex differences in motivational circuitry and behavior suggest that females may be more sensitive to rewarding stimuli. These differences have been implicated for the observed vulnerability in women to substance use disorders. METHODS: We used an optogenetic self-stimulation task in addition to ex vivo electrophysiological recordings of NAcSh neurons in mouse brain slices to investigate potential sex differences in ILC-NAcSh circuitry in reward-seeking behavior. Glutamatergic neurons in the ILC were infected with an AAV delivering DNA encoding for channelrhodopsin. Entering the designated active corner of an open field arena resulted in photostimulation of the ILC terminals in the NAcSh. Self-stimulation occurred during two consecutive days of testing over three consecutive weeks: first for 10 Hz, then 20 Hz, then 30 Hz. Whole-cell recordings of medium spiny neurons in the NAcSh assessed both optogenetically evoked local field potentials and intrinsic excitability. RESULTS: Although both sexes learned to seek the active zone, within the first day, females entered the zone more than males, resulting in a greater amount of photostimulation. Increasing the frequency of optogenetic stimulation amplified female reward-seeking behavior. Males were less sensitive to ILC stimulation, with higher frequencies and repeated days required to increase male reward-seeking behavior. Unexpectedly, ex vivo optogenetic local field potentials in the NAcSh were greater in slices from male animals. In contrast, female medium-spiny neurons (MSNs) displayed significantly greater intrinsic neuronal excitability. CONCLUSIONS: Taken together, these data indicate that there are sex differences in the motivated behavior driven by glutamate within the ILC-NAcSh circuit. Though glutamatergic signaling was greater in males, heightened intrinsic excitability in females appears to drive this sex difference.

The shell region of the nucleus accumbens (NAcSh) is involved in motivation and reward. It receives excitatory glutamatergic inputs from multiple brain regions. One specific region is the infralimbic cortex (ILC), which when activated, influences reward-seeking behavior. While previous research has focused on males, there are inherent sex differences in reward circuitry and reward-seeking behavior. Using an optogenetic self-stimulation task, in addition to ex vivo electrophysiological recordings, we found inherent sex differences in the ILC-NAcSh circuit in behavioral output, synaptic strength, and intrinsic neurophysiology. Female mice showed more robust reward-seeking behavior. Increasing the frequency of stimulation intensified this behavior in females, while males required higher frequencies and repeated testing days to increase their reward-seeking behavior. Surprisingly, optogenetically stimulating the ILC terminals in the NAcSh in brain slices resulted in stronger responses in males. More consistent with the behavioral data, female MSNs displayed higher intrinsic excitability. Our results suggest that there are sex differences in motivated behavior, driven by glutamatergic signaling in the ILC-NAc circuit. Despite stronger ILC-based glutamatergic signaling in males, heightened intrinsic excitability of MSNs in females seems to be the driving force behind this sex difference in reward-seeking behavior. These findings contribute to our understanding of the neural mechanisms behind sex-based differences in motivation and their potential implications for substance use disorders.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

A quadruple dissociation of reward-related behaviour in mice across excitatory inputs to the nucleus accumbens shell.

The nucleus accumbens shell (NAcSh) is critically important for reward valuations, yet it remains unclear how valuation information is integrated in this region to drive behaviour during reinforcement learning. Using an optogenetic spatial self-stimulation task in mice, here we show that contingent activation of different excitatory inputs to the NAcSh change expression of different reward-related behaviours. Our data indicate that medial prefrontal inputs support place preference via repeated actions, ventral hippocampal inputs consistently promote place preferences, basolateral amygdala inputs produce modest place preferences but as a byproduct of increased sensitivity to time investments, and paraventricular inputs reduce place preferences yet do not produce full avoidance behaviour. These findings suggest that each excitatory input provides distinct information to the NAcSh, and we propose that this reflects the reinforcement of different credit assignment functions. Our finding of a quadruple dissociation of NAcSh input-specific behaviours provides insights into how types of information carried by distinct inputs to the NAcSh could be integrated to help drive reinforcement learning and situationally appropriate behavioural responses.

Cancer treatment induces neuroinflammation and behavioral deficits in mice.

Introduction: Cancer survivors are increasingly diagnosed with a syndrome of neurocognitive dysfunction termed cancer-related cognitive impairment (CRCI). Chemotherapy and radiation therapy have been implicated in CRCI; however, its underlying pathogenesis remains unclear, hindering effective prevention or treatment. Methods: We used the hairless strain SKH1 (11-12-week-old) and treated the mice with radiation to the right hindlimb, doxorubicin (a chemotherapy agent), concurrent radiation, and doxorubicin, or no treatment (control). Neurocognition was evaluated via standardized behavioral testing following treatment. Mice were subsequently humanely euthanized, and plasma and brains were collected to identify inflammatory changes. Results: Mice treated with radiation, doxorubicin, or both radiation and doxorubicin demonstrated equivalent hippocampal dependent memory deficits and significant increases in activated microglia and astrocytes compared to control mice. Doxorubicin-treated mice had significantly increased plasma IL-6 and failed to gain weight compared to control mice over the study period. Discussion: This study demonstrates that non-brain directed radiation induces both gliosis and neurocognitive deficits. Moreover, this work presents the first characterization of SKH1 mice as a relevant and facile animal model of CRCI. This study provides a platform from which to build further studies to identify potential key targets that contribute to CRCI such that strategies can be developed to mitigate unintended neuropathologic consequences associated with anticancer treatment.

Na+/H+ antiport is essential for Yersinia pestis virulence.

Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH.

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K(+) in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K(+) or Na(+) ions for H(+) within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K(+)(Na(+))/H(+) antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment.

The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo.

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.

Virulence of metalloproteases produced by Vibrio species on Pacific oyster Crassostrea gigas larvae.

Vibrio tubiashii, a pathogen of shellfish larvae and juveniles, produces several extracellular products. Here, we document that culture supernatants of several marine Vibrio species showed toxicity to oyster larvae. Treatment of these supernatants with EDTA not only severely diminished proteolytic activities, but also dramatically reduced toxicity to the larvae. Culture supernatants of metalloprotease-deficient mutants of V. tubiashii, V. cholerae, and V. splendidus were impaired in their ability to cause larval death compared to the wild type strains. Culture supernatants of Pseudomonas aeruginosa, known to contain several secreted proteases, showed virtually no toxicity to oyster larvae. Purified V. tubiashii protease A (VtpA), but not the prototype metalloprotease, thermolysin from Bacillus thermoproteolyticus, was highly toxic to the larvae. In addition, toxicity of purified VtpA was much greater for 6-d-old oyster larvae than for 16-d-old larvae. Together, these results indicated that culture supernatants of a variety of Vibrio species are highly toxic to oyster larvae and that the production of a metalloprotease is required for this effect. We propose that there are, as yet uncharacterized, specific substrates contained in larval tissue that are degraded by VtpA as well as certain homologous metalloproteases produced by other marine Vibrio species which, in turn, may contribute to vibriosis.

The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for pacific oyster (Crassostrea gigas) larvae.

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.