Two Metalloproteases VdM35-1 and VdASPF2 from Verticillium dahliae Are Required for Fungal Pathogenicity, Stress Adaptation, and Activating Immune Response of Host

ABSTRACT Verticillium dahliae is a soilborne fungus that causes destructive vascular wilt diseases in a wide range of plant hosts. In this study, we identified two M35 family metalloproteinases: VdM35-1 and VdASPF2, and investigated their function in vitro and in vivo. The results showed that VdM35-1 and VdASPF2 were located in the cell membrane, as secreted proteins depended on signal peptide, and two histidine residues (H) induced cell death and activated plant immune response. VdM35-1 depended on membrane receptor proteins NbBAK1 and NbSOBIR1 in the process of inducing cell death, while VdASPF2 did not depend on them. The deletion of VdM35-1 and VdASPF2 led to the decrease of sporulation and the slow shortening of mycelial branch growth, and the spore morphology of VdM35-1-deficient strain became malformed. In addition, ΔVdM35-1 and ΔVdASPF2 showed more sensitive to osmotic stress, SDS, Congo red (CR), and high temperature. In terms of the utilization of carbon sources, the knockout mutants exhibited decreased utilization of carbon sources, and the growth rates on the medium containing sucrose, starch, and pectin were lower than the wild type strain, with significantly limited growth, especially on galactose-containing medium. Furthermore, ΔVdM35-1 and ΔVdASPF2 showed a significant reduction in pathogenicity. Collectively, these results suggested that VdM35-1 and VdASPF2 were important multifunction factors in the pathogenicity of V. dahliae and relative to stress adaptation and activated plant immune response. IMPORTANCE Verticillium wilt, caused by the notorious fungal pathogen V. dahliae, is one of the main limiting factors for agricultural production. Metalloproteases played an important role in the pathogenic mechanism of pathogens. Our research found that M35 family metalloproteases VdM35-1 and VdASPF2 played an important role in the development, adaptability, and pathogenicity of V. dahliae, providing a new perspective for further understanding the molecular mechanism of virulence of fungal pathogens.

Therefore, in-depth understanding of the molecular pathogenic mechanism of V. dahliae is beneficial to control Verticillium wilt of cotton.
The plant immune response is mainly composed of two layers of defense response in plants (4). The first layer defense system is a disease-resistant response triggered by pattern recognition receptors (PRRs) on the plant cell membrane surface that recognize the conserved pattern molecules of pathogenic microorganisms (microbe-associated molecular patterns [MAMPs] or pathogen-associated molecular patterns [PAMPs]), which is called PTI (PAMP-triggered immunity) (5). The second layer of defense response is that the nucleotide-binding leucine-rich repeat (NB-LRR) type of disease-resistant proteins in plant cells directly or indirectly identify the effectors secreted by pathogenic microorganisms into plant cells, triggering a stronger immune response, which is called effector-triggered immunity (ETI) (6). Plant PTI, also known as basic resistance, is a series of reactions through which plants perceive PAMPs of pathogenic microorganisms through receptor-like kinases (RLKs) or receptor-like proteins (RLPs) on the cell membrane surface, thereby triggering plant cells (7). For example, the effector proteins Vd424Y, VdEG1, and VdXyn4 secreted by V. dahliae were recognized by the core immune receptors on the plasma membrane of plants, which triggered the immune response of plants and upregulated the expression of disease-related genes PRs (8)(9)(10). Effector proteins secreted by pathogens inhibit plant immune responses in many ways, including PTI inhibition (11), so that pathogens can infect plants successfully (12)(13)(14)(15). In order to resist the pathogenic microorganism, host cells trigger ETI by recognizing effectors (16). ETI often produces large amounts of peroxides (such as reactive oxygen species [ROS]) in infected plant cells and induces hypersensitive response (HR) in plants (16).
Effector proteins play an important role in the process of pathogen infection. Most effector proteins have conserved motifs at the N or C terminus, such as RXLR, CRN, CFEM, RGD, DELD, RYWT, or Y/F/WXC (3,17,18). V. dahliae can secrete large amounts of effector proteins into the host. For example, VdNEP, PevD1, or VdCP1 can induce plant cell death and trigger immune response (9,19,20). In addition, metalloproteases play important roles in the pathogenic mechanisms of pathogens, which were widely distributed in many pathogens such as V. dahliae and Fusarium oxysporum (21). Among them, the zinc ion-dependent metalloprotease contained conserved HEXXH domain, and two histidine residues were used as the first and second zinc ligands, respectively (22). F. oxysporum metalloprotease FoMep1 and serine protease FoSep1 had a synergistic effect on the chitinase cleavage of tomato, thereby reducing antifungal activity and enhancing virulence of pathogens (23). Most M35 families belong to zinc ion-dependent metalloproteases secreted by pathogens. Avr-Pita of the M35 family of Magnaporthe grisea proved to be an effector protein interacting with Pi-ta in plant cells to trigger host defense response (24). Rhizoctonia cerealis M35 family RcMEP2 and an entomopathogenic fungus Metarhizium anisopliae M35-4 had secretory activity and played a role in host infection as pathogenic factors (25,26). Previous studies have shown that V. dahliae VdASPF2 secreted protein with M35 family conserved domain. VdASPF2 induced microsclerotia production and melanin accumulation under nutrient stress and low-temperature conditions, resulting in the decrease of pathogenicity of the deletion mutant to cotton (27).
In this study, a metalloprotease VdM35-1 containing the conserved domain of M35 family was identified in V. dahliae, which contained a signal peptide. The conserved motif HEXXH in VdM35-1 was similar to the conserved motif HRXXH of the published zinc iondependent metalloprotease VdASPF2 (28). Both could induce cell death in Nicotiana benthamiana leaves and were mainly located on the cell membrane and induced cell death depending on two histidine residues and signal peptides. Cell death induced by VdM35-1 was dependent on BAK1 and SOBIR1, while the cell death induced by VdASPF2 was independent of BAK1 and SOBIR1. In addition, DVdM35-1 and DVdASPF2 mutants exhibited more sensitivity to potassium chloride (KCl), sodium chloride (NaCl), sorbitol, SDS, Congo red (CR), and high temperatures. These results of different carbon source utilization experiments showed that the utilization capacities of DVdM35-1 and DVdASPF2 for sucrose, raffinose, and starch were decreased, especially on galactose-containing medium. Disease symptom observations showed that the virulence of deletion mutants to cotton was significantly reduced compared with that of the wild type strains. Collectively, these results showed that metalloproteases VdM35-1 and VdASPF2 positively regulated the virulence of V. dahliae and played an important role in the normal nutritional growth and stress response.

RESULTS
Identification of two secreted proteins from V. dahliae that induced plant cell death. A metalloprotease (VDAG_00129) was identified in the V. dahliae strain VdLs17 (https://www.ncbi.nlm.nih.gov/genome/832), which had a conserved motif of M35 family and domain HEXXH named VdM35-1. In the previous reports, VdASPF2 (VDAG_04551) containing the conserved domain of M35 family also had a similar conserved motif HRXXH (27, 28) (Fig. 1A). The full-length cDNA of VdM35-1 and VdASPF2 was cloned from the strong pathogenic defoliating strain Vd080. The N-terminal signal peptides of VdM35-1 and VdASPF2 were predicted by SignaIP 5.0, and the yeast signal trap system assay showed that YTK12-pSUC2-VdM35-1SP and VdASPF2SP exhibited the normal growth on CMD-W and YPRAA medium, which suggested that VdM35-1 and VdASPF2 were most likely secreted into the extracellular space during infection (Fig. 1B). Furthermore, the subcellular localization in plants of the two candidate effectors showed that VdM35-1 (full-length amino acid sequence) and VdASPF2 (full-length amino acid sequence) were mainly located on the cell membrane of plants ( Fig.  S1A and B). Their localization on plant cell membrane were further verified by onion plasmolysis (Fig. S1C). Then, cell death experiments were performed in N. benthamiana leaves. The apoptosis-inducing related proteins (BAX and INF1) and empty vector green fluorescent protein (GFP) were used as positive and negative controls, respectively. The results showed that both VdM35-1 and VdASPF2 could induce cell death (Fig. 1C). Both candidates were confirmed to be expressed in N. benthamiana by Western blotting (Fig. 1D). The electrolyte leakage around the VdM35-1 and VdASPF2 expression regions were significantly increased compared to the GFP expression region. It was found that both VdM35-1 and VdASPF2 could trigger the burst of ROS (Fig. S2). These results suggested that both VdM35-1 and VdASPF2 could induce cell death.
VdM35-1 contained the conserved motif HEXXH, while VdASPF2 had the conserved motif HRXXH. To determine whether VdM35-1 and VdASPF2-induced cell death   Fig. 2A). All four candidates were confirmed to be expressed in N. benthamiana by Western blotting (Fig. 2B). The results showed that VdM35-1-1 and VdASPF2-1 did not induce cell death, whereas expression of both VdM35-1-2 and VdASPF2-2 could slightly induce cell death (Fig. 2C). For detection of H 2 O 2 production and electrolyte leakage in N. benthamiana, all results were consistent with the phenotype (Fig. S2A and B). These results showed that VdM35-1 and VdASPF2 required two histidine residues and SP to induce cell death.
VdM35-1 and VdASPF2 triggered plant immunity response. HR is a type of programmed cell death that is generally considered to be closely related to the plant defense response. To determine whether VdM35-1 and VdASPF2-activated plant immune response, we examined a HR-specific marker gene (NbHIN1) in N. benthamiana leaves following VdM35-1 and VdASPF2 infiltration via Agrobacterium. The results showed that NbHIN1 was significantly activated by VdM35-1 and VdASPF2 at 3 days after infiltration, which indicates that VdM35-1 and VdASPF2 could trigger HR-associated immunity of N. benthamiana. Quantitative reverse transcription-PCR (RT-qPCR) was used to detect the SA-dependent immunity marker genes NbPR2, NbPR1a, and NbLOX. The relative expression of these genes increased significantly ( Fig. 3A). Therefore, VdM35-1 and VdASPF2 could induce plant immune response by activating SA-mediated defense pathways.
Because VdM35-1 and VdASPF2 could induce apoptosis and trigger plant immune response, we speculated that VdM35-1 and VdASPF2 may play a role as a PAMP. Therefore, the relative expression of PTI marker genes was measured in this experiment. The results showed that the relative expression levels of PTI marker genes NbWRKY7, NbWRKY8, NbACRE31, and NbPTI5 were significantly increased after VdM35-1 and VdASPF2 were infected by Agrobacterium (Fig. 3B), respectively, indicating that VdM35-1 and VdASPF2 may be PAMPs, and they participated in plant-induced immune response through a PTI process. VdM35-1, rather than VdASPF2, induced cell death in N. benthamiana depending on NbBAK1 and NbSOBIR1. BAK1 and SOBIR1 are membrane receptor-associated kinases involved in immune responses triggered by some PAMPs in plants. In order to verify whether NbBAK1 and NbSOBIR1 mediate VdM35-1 and VdASPF2 induced cell death in N. benthamiana, virus-induced gene silencing (VIGS) was performed to silence NbBAK1 and NbSOBIR1 in N. benthamiana leaves, and RT-qPCR was used to confirm the silencing of NbBAK1 and NbSOBIR1 at 21 days after VIGS-mediated gene silencing (Fig. 4B). Then, VdM35-1, VdASPF2, and BAX were transiently expressed in N. benthamiana leaves of silenced NbBAK1 and NbSOBIR1, and the expression of VdM35-1 and VdASPF2 was confirmed by Western blotting analysis (Fig. 4C). The results showed that BAX and VdASPF2 retained their ability to induce cell death. In contrast, VdM35-1 no longer induced cell death (Fig. 4A). These results suggested that VdM35-1-induced cell death was dependent on NbBAK1 and NbSOBIR1, whereas VdASPF2-induced cell death was independent of NbBAK1 and NbSOBIR1.

Functions of Two Metalloproteases in V. dahliae
Microbiology Spectrum strains on PDA were lower than the wild type strain ( Fig. 5D and E). The observation of spore morphology showed that DVdM35-1 led to spore malformation and presented the shape of a concave disk, while the spore morphology of DVdASPF2 was similar to the wild type strain (Fig. 5B). The hyphal round branch ends of DVdM35-1 and DVdASPF2 were shorter than those of the wild type strains (Fig. 5F).
In order to detect the ability of DVdM35-1 and DVdASPF2 to utilize different carbon sources, the growth rates were monitored on the observation medium with sucrose, galactose, raffinose, and starch as carbon sources. The wild type strain Vd080 grew well in all

Functions of Two Metalloproteases in V. dahliae
Microbiology Spectrum carbon source tests. The growth rate of knockout mutants on medium containing sucrose and starch were lower than that of wild type strains (Fig. S4). Furthermore, the growth of DVdM35-1 and DVdASPF2 on galactose-containing medium were significantly limited, with colony diameters of 50 and 47% of wild type strains, respectively (Fig. S4). These results showed that under normal growth conditions, the deletion of VdM35-1 and VdASPF2 not only led to the decrease of conidial yield but also affected mycelial growth and reduced the ability of V. dahliae to utilize different carbon sources. VdM35-1 and VdASPF2 participated in stress adaptation. In order to detect the response of DVdM35-1 and DVdASPF2 to different stresses, the growth rates were monitored on the PDA medium containing KCl, NaCl, sorbitol, SDS, and CR, respectively. The colony diameters of DVdM35-1 and DVdASPF2 were smaller and more sensitive to stress (Fig. 6A to C). The growth of wild type strain Vd080 on PDA plates containing SDS and CR were similar to that on normal PDA plates, but the growth of DVdM35-1 and DVdASPF2 were inhibited, and the colony diameters were 56, 67, 71, and 66% that of wild type strains, respectively ( Fig. 4A and 5A). DVdM35-1 was more sensitive to sorbitol and SDS stress than DVdASPF2. After the spores of each strain were treated at 45 and 4°C for an hour, spore germination was observed at different time intervals. After high-temperature treatment, spores of wild type strain Vd080 germinated and began to form hyphae at 6 h, while spores of DVdM35-1 and DVdASPF2 germinated and began to form hyphae at 12 h (Fig.  S5A). Compared with the wild type strain Vd080, it was found that high-temperature treatment delayed the spore germination of DVdM35-1 and DVdASPF2. The time of spore germination after low-temperature treatment was the same as wild type strain Vd080 (Fig.  S5B). After 3 min of UV irradiation, the spore germination of each strain was observed at different time points. It was found that, like the wild type strain Vd080, the spores of DVdM35-1 and DVdASPF2 germinated at 6 h and began to form hyphae. At 12 h, hyphae formed branches near the spores (Fig. S5B). These results showed that under stress conditions, the deletion of VdM35-1 and VdASPF2 would lead to more sensitivity of V. dahliae to stress.
VdM35-1 and VdASPF2 were not involved in mycelium penetration. In order to detect the penetration ability of DVdM35-1 and DVdASPF2 on cellophane, equal amounts of conidia of each strain were coated on cellophane laid on PDA medium for 3 days. The results showed that all strains could grow normally on cellophane (Fig. 7A). Three days after removing the cellophane, DVdM35-1 and DVdASPF2 were observed to grow. Observation of mycelium growth on cellophane showed that the mycelium growth of the knockout mutants was sparse and chaotic, while the mycelium of wild type strain and complemented strain was uniform and normal (Fig. 7B). Conidia of all strains were observed on the back of the cellophane (Fig. 7C). These results indicated that VdM35-1 and VdASPF2 did not affect the penetration of mycelium.
VdM35-1 and VdASPF2 positively regulated pathogenicity. During the infection of cotton by V. dahliae, the expression levels of VdM35-1 and VdASPF2 in the early stage were significantly increased (Fig. S6). In order to detect the roles of VdM35-1 and VdASPF2 in virulence, the wild type, knockout mutants, and complemented mutants were used for pathogenicity tests on susceptible upland cotton Jimian11. The results showed that the seedlings of cotton wilted, yellowed, and even dropped leaves after inoculation with conidia suspension of wild type strain and complementation mutant strain. After inoculation with DVdM35-1 or DVdASPF2 conidial suspension, cotton seedlings showed slight wilting and yellowing (Fig. 8A) (Fig. 8D). The knockout mutants had slight more browning of the vascular bundle than wild type and complemented mutants, but the vascular bundle browning degree of plants by DVdASPF2 infection was more serious than that of DVdM35-1 infection (Fig. 8C). The fungal recovery test verified that each strain caused plant disease after infection (Fig. 8B). According to the fungal setting values, the fungal biomass of

Functions of Two Metalloproteases in V. dahliae
Microbiology Spectrum knockout mutants were lower than those of wild type and complemented mutants. In addition, compared with wild type Vd080, the colonization of DVdM35-1 and DVdASPF2 strains in cotton roots was also reduced (Fig. 8E). These results suggest that VdM35-1 and VdASPF2 positively regulate the virulence of V. dahliae and indicate that VdM35-1 played a more important role than VdASPF2 in the virulence of V. dahliae.

DISCUSSION
V. dahliae is a vascular fungal pathogen, which can destroy the vascular bundle of host cells and cause damage to plants (2,29). Relatively few secretory effector proteins were found to play a role in virulence. Metalloproteinases are considered important virulence factors in fungal pathogens (30,31), which are widely distributed as in V. dahliae, F. oxysporum, and other pathogens (21). Previous studies found that M35 family metalloproteinases had a conserved motif of HEXXH, which were zinc-dependent metalloproteinases and played an important role in the interaction between pathogens and hosts (32). However, the function of M35 family metalloproteases in V. dahliae were still unclear. In this study, a metalloprotease VdM35-1 containing the conserved motif of HEXXH and one metalloprotease VdASPF2/VDAL containing the conserved motif of HRXXH were found (Fig. 1A). Previous studies showed that VdASPF2 contained the conserved motif of HRXXH (27) and had the binding activity of zinc ions (28). Both VdM35-1 and VdASPF2 had the ability to induce the plant immunity of cell death by targeting plant explants. Studies have shown that many key interactions between plants and pathogens play a role in the space of apoptosis (33,34). Like PAMP, Vd424Y of V. dahliae and FoEG1 of F. oxysporum could induce cell death, and their signal peptides were required. When triggering plant defense response, the related genes were upregulated (8,35). FocM35-1 identified from F. oxysporum can trigger the accumulation of ROS when inducing cell death (32). This study found that VdM35-1 and VdASPF2 had similar functions and lost the ability to induce cell death after the signal peptide was truncated. In addition, subcellular localization showed that VdM35-1 and VdASPF2 were located in plant cell membrane (Fig. S1). These results showed that the extracellular space was very important for the functions of VdM35-1 and VdASPF2. Previous studies have shown that membrane metalloproteinase plays a role in many signaling pathways (36). VdM35-1 and VdASPF2 played roles in cell membrane through plastids and could trigger plant defense responses, including ROS accumulation. (Fig. S2). VdM35-1 and VdASPF2 significantly activated the upregulation of HR (NbHIN1) (37), SA (NbPR2, NbPR1a, and NbLOX) (35,38), and PTI (NbWRKY7, NbWRKY8, NbACRE31, and NbPTI5) (8,35,(38)(39)(40) related marker genes, respectively. (Fig. 3). Previous studies have found that M35 and M36 family metalloproteases contain conserved motif HEXXH, which is a zinc-dependent metalloprotease, and it has been confirmed that they contain zinc-binding activity of VDAL/VdASPF2 from V. dahliae (28,41). Further, the truncated conserved motifs lost the ability to induce cell death (Fig. 2). These results showed

Functions of Two Metalloproteases in V. dahliae
Microbiology Spectrum that VdM35-1 and VdASPF2 were death-inducing proteins through exosomes to cell membrane. The functions of VdM35-1 and VdASPF2 mainly depended on the membrane localization of host cells. Previous studies have shown that fungal effectors trigger plant defense responses through the perception of cell membrane immune receptors BAK1 and SOBIR1 (33,42). For example, the secretion proteins VdEG1, VdEG3, and Vd424Y of V. dahliae induced cell death in N. benthamiana through BAK1/SOBIR1-mediated signaling pathways (8,9). However, VdRTX1-mediated plant cell death was independent of BAK1 and SOBIR1 (43). Silencing BAK1 or SOBIR1 genes in this formula showed that VdM35-1-induced plant cell death depended on BAK1 and SOBIR1, while VdASPF2-induced plant cell death was independent of BAK1 and SOBIR1. These results suggested that VdM35-1 may be an ectoplast effector, while VdASPF2 is not (Fig. 4). Although they both had M35 family metalloproteinases domains, they induced plant immunity in different ways.
Mycelium and conidia play important roles in the growth cycle of V. dahliae (44). Normal vegetative growth of V. dahliae is regulated by many genes. Previous studies have shown that the absence of VdSOD1 and VdSOD3 in V. dahliae does not affect sporulation, spore morphology, and normal vegetative growth of mycelium (45,46). Conidia of the DVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches (47). In this study, it was found the sporulation decreased, and the mycelial branch growth became shorter in VdM35-1 and VdASPF2 deletion mutants (Fig. 5D and E and F). Especially, spore morphology of the DVdM35-1 was changed (Fig. 5B). Previous studies have found that deletion of VdHP1 could also lead to changes in spore morphology and improve the utilization of different carbon sources to maintain the normal vegetative growth (48). However, the ability of V. dahliae to utilize different carbon sources decreased to various degrees when VdM35-1 and VdASPF2 were deleted (Fig. S4). Interestingly, VdM35-1 and VdASPF2 did not affect the ability of V. dahliae to penetrate cellophane (Fig. 7). These results showed that VdM35-1 and VdASPF2 were important factors involving in the vegetative growth of V. dahliae.
Previous studies had shown that VdHog1, VdPbs2, and VdSsk2 were highly responsive to osmotic stress (44,49,50). VdUGP and VdSkn7 showed high sensitivity to SDS and CR stress. VdHP1 could enhance the resistance of V. dahliae to UV and high temperature and played an important role in the pathogenicity of pathogens (48). In this study, VdM35-1 and VdASPF2 were found to be involved in regulating the responses of V. dahliae to osmotic stress NaCl, KCl, and sorbitol. DVdM35-1 and DVdASPF2 were sensitive to osmotic stress, SDS, and CR stress, with growth retardation, thereby reducing its pathogenicity (Fig. 6), which indicated that there were defects in the cell walls of DVdM35-1 and DVdASPF2, leading to the decrease of its pathogenicity. Collectively, these results showed that VdM35-1 and VdASPF2 may be involved in various stresses of V. dahliae to the environment, helping pathogenic fungi survive in nature.
The metalloprotease is also involved in the regulation of pathogenicity. Previous studies have shown that FocM35_1, RcMEP2-RcMEP5, and MrM35-4 played important roles in pathogenicity (25,26,32). After gene deletion, the pathogenicity of deletion mutants was significantly decreased compared to the wild type. In this study, it was found that after inoculation of cotton with conidial suspensions of DVdM35-1 and DVdASPF2 mutants, the incidence and severity of cotton verticillium wilt were significantly reduced compared to wild type and complemented mutants. By investigating the disease index, measuring fungal biomass, and observing vascular browning, these results showed that the pathogenicity of the knockout mutants were significantly decreased, but the pathogenicity of DVdASPF2 was stronger than DVdM35-1 (Fig. 8). It is concluded that VdM35-1 and VdASPF2 were positive regulators of virulence and related to the pathogenicity of V. dahliae.
In summary, this study demonstrated that VdM35-1 and VdASPF2 could effectively activate the immune response of plants and regulate the pathogenicity of V. dahliae by participating in vegetative growth, conidia production, and mycelial growth, which suggest that metalloproteinases play an important role in the development, adaptability, and pathogenicity of V. dahliae, and provided a new perspective for further understanding the molecular pathogenicity mechanism of fungal pathogens. Further research is necessary to elucidate the VdM35-1 and VdASPF2 signaling mechanism in V. dahliae and to search for interacting proteins in cotton.

MATERIALS AND METHODS
Strains and plants. The wild type V. dahliae strain Vd080 is a strong pathogenic defoliating strain and is cultured in liquid Czapek Dox medium or on potato dextrose agar medium (48). Upland cotton (Gossypium hirsutum) cultivar Jimian11 was highly susceptive to V. dahliae and was grown in a 28°C temperature-controlled glasshouse (14-h light/10-h dark). N. benthamiana was grown in the growth chamber at 25°C.
Yeast signal sequence trap system. The yeast signal peptide screening was used to verify the secretory function of effector protein signal peptides (51). The yeast strain chosen for the experiment was YTK12 (52); it lacked the sucrose convertase gene and cannot grow on a medium with sucrose as the only carbon source. The predicted signal peptide was inserted into the vector pSUC2. Transfer of recombinant plasmid to yeast strain YTK12 screened positive clones in CMD-W medium (deletion of tryptophan). Positive clones grown on CMD-W medium were transferred to YPRAA machine (2% raffinose) and incubated upside down at 28°C. The function of signal peptide was screened and verified by YPRAA medium and TTC coloration test. Further, the signal peptide function was validated by chromogenic reaction. The primers used in this assay are listed in Table S1.
VIGS in N. benthamiana. In TRV-mediated gene silencing assays, the plasmid constructs pTRV1, pTRV2::NbBAK1, pTRV2::NbSOBIR1, pTRV2, and pTRV2::PDS were introduced into A. tumefaciens GV3101 (53,54). The two primary leaves of four-leaf-stage N. benthamiana seedlings were injected with a mixture (1:1 ratio) of A. tumefaciens culture (OD 600 = 1.8) containing pTRV1 and pTRV2::genes. The pTRV2:: PDS was used as a positive control, while the pTRV2 served as a control (58,59). The RNA extracted from leaves was used to validate the efficiency of gene silencing by RT-qPCR. The primers used in this assay are listed in Table S1.
Subcellular localization analysis. The recombinant plasmids 35S::VdM35-1 and 35S::VdASPF2 were transformed into A. tumefaciens GV3101, respectively. The resulting strains were expressed in 4-week-old leaves of N. benthamiana using the previously described method (53,54). After 48 h of Agrobacterium infiltration, green fluorescence of enhanced GFP (eGFP) was detected by 488-nm confocal microscopy. Using biolistic technology, high-pressure gas was used to accelerate the gold powder particles wrapped with recombinant plasmids directly into the onion skin. After 48 h, green fluorescence of eGFP was detected by confocal microscopy. The protoplasts of Arabidopsis thaliana were extracted, and the recombinant plasmids were transformed using PEG-mediated method. After 16 h of treatment under low light, the fluorescence was observed with a laser confocal microscope. The primers used in this assay are listed in Table S1.
Target gene knockout and complementation. Knockout and complemented mutants were obtained by the Agrobacterium-mediated method (60,61). The upstream fragment (Up) ;1-kb and downstream fragment (Down) ;1-kb sequences of VdM35-1 and VdASPF2 gene were selected. The upstream and downstream fragments corresponding to each gene were amplified from Vd080 genomic DNA. The hygromycin (Hyg) fragment was amplified in vector B303. These fragments were fused to linearized B303 using recombinant enzymes (ClonExpress Ultra one step cloning kit; Vazyme, Nanjing, China) to construct the vector Up-Hyg-Down-B303-Hyg according to the manufacturer's instructions. The constructed recombinant plasmid was transformed into Agrobacterium GAL1 and then transformed into V. dahliae. The positive colonies were screened on PDA containing hygromycin and confirmed by PCR with the primers Hyg-F/R. The copy of Hyg was confirmed by Southern blotting. The recombinant plasmid Up-VdM35-1/VdASPF2-Down-pCAMBIA1302 were obtained by the same method. The recombinant vector was transformed into A. tumefaciens and then cotransformed into the knockout strain to screen the positive transformants of the complemented mutants. The primers used in this assay are listed in Table S1.

Functions of Two Metalloproteases in V. dahliae
Microbiology Spectrum Phenotypic analysis of V. dahliae mutants. The wild type, knockout, and complemented mutant strains of V. dahliae were inoculated in the liquid Czapek Dox medium and cultured at 25°C for 5 days, and the conidial concentrations of each strain were adjusted to be the same. 5 mL conidia were taken and inoculated on a uniform and fresh PDA plate. The plate was placed in a 25°C incubator for inverted culture for 14 days, and the colony diameters were measured and photographed. Each strain was set at least three replicates (48).
In 5-mm colonies, the wild type, knockout, and complemented mutant strains of V. dahliae were obtained on the plate and inoculated on PDA plates. After 3 days of incubation at 25°C, the sterilized coverslips were obliquely inserted into the medium at the edge of the colony at 45°C for 24 h, and then the coverslips were taken out to observe the mycelial morphology of different strains under a stereo microscope (35).
The wild type, knockout, and complemented mutants strains of V. dahliae were inoculated on PDA plates and cultured at 25°C for 7 days. Fungal colonies with diameters of 5 mm were obtained on the plates with a sterile puncher. The strains were inoculated in a liquid Czapek Dox Medium. One fungal cake was inoculated in each bottle and cultured at 25°C for 3 days at 180 r/min. The filtrate was filtered and collected with a sterile gauze. Then, the concentration of conidia was counted under a microscope with a blood cell count plate. Each strain was set with at least three replicates (35). The conidial solution was taken to prepare frozen sections, and the spore morphology was observed by scanning electron microscopy for photographing.
Carbon source utilization assays. In order to analyze the utilization of carbon sources of wild type, knockout, and complemented mutants, galactose (10 g/liter), sucrose (30 g/liter), raffinose (10 g/liter), and starch (17 g/liter) were added to Czapek Dox medium with sucrose deficiency, respectively. The experiment was repeated three times.
Growth of mutants under stress treatments. In order to detect the sensitivity of wild type, knockout, and complemented mutants strains to cell wall inhibitors and osmotic stress, all strains were cultured in PDA medium containing 1 M NaCl, 1 M KCl, 1 M sorbitol, 0.02% CR, and 0.002% SDS (44,62,63). The diameter was measured after 14 days of incubation at 25°C. Each strain was set at least three replicates.
In order to determine the thermal stability and UV treatment, the conidia (1 Â 10 7 CFU/mL) of all strains were collected from Czapek liquid medium for 5 days. 1 mL suspension was treated at 45 or 4°C with UV light for 1 h, 1 h, or 3 min (48), respectively. Then, the suspension was cultured on PDA at 25°C for 0, 6, and 12 h. The germination was observed under microscope. Each strain was set with at least three replicates.
RT-qPCR analysis. Total RNA was isolated using polysaccharide polyphenol plant RNA extraction kit (RC411-C1), quantified, and used as a template for reverse transcription with the HiScript II QRT SuperMix for qPCR (1gDNA wiper R223-01) (Vazyme). The RT-qPCR assays were performed using the ChamQ universal SYBR qPCR Master Mix (Q711-02) (64). The NbActin gene was used as internal control. Three independent biological and technical repeats were performed.
Mycelial penetration assays. The conidia were coated on cellophane to observe the difference of penetration ability of different strains. The morphology of mycelium on glass paper was observed by scanning electron microscope (48,68). The experiment was repeated three times.
Protein extraction and Western blotting. The protein was extracted from leaves of 4-week-old N. benthamiana after Agrobacterium infestation. After 36 h of infestation, equal-weight leaves were ground in liquid nitrogen and added with the corresponding volume of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime P0013C). The mixture was lysed on ice for 15 min, then centrifuged at 13,000 Â g for 10 min at 4°C, transferred to a new tube, and hyperinactivated by adding protein buffer to the collected supernatant solution for 5 min. Protein analysis by SDS-PAGE and electroblotting onto polyvinylidene difluoride (PVDF) membranes (69,70).
Southern blotting. The genomic DNA of the wild type, DVdM35-1-1, DVdM35-1-2, DVdASPF2-1, and DVdASPF2-2 were digested by HindIII, then separated by 0.8% agarose gel electrophoresis, and transferred to the Hybond N 1 nylon membrane. The cDNA of Hyg was labeled with digoxin as a probe for hybridization. The specific steps were carried out according to the DIG high prime DNA labeling and detection starter kit I instructions (Roche) using the transfer membrane method with reference to Southern blotting (48,71).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1.1 MB.


Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Introduction
Amyotrophic lateral sclerosis (ALS) derives its name from denervational atrophy of the muscles in the tongue, oropharynx, and limbs (amyotrophy), degenerating corticospinal axons resulting in thinning and scarring (sclerosis) of the lateral spinal cord, and thinning of the ventral roots. ALS is a progressive, paralytic disease due to loss of motor neurons (MN) in the brain and spinal cord. ALS signs include diffuse muscle weakness, fasciculations (spontaneous muscle twitching), hyperreflexia (upper), and paralysis (lower), and ultimately reducing life expectancy to 2-5 years [1,2]. In addition to these motor-associated pathologies, 15-20% of ALS cases also exhibit cognitive deficits as a result of degeneration of the frontal and temporal lobes (frontotemporal dementia or FTD) [3]. Incidence of ALS is 3-5 per 100,000 persons globally and is present in both dominantly inherited familial (FALS, 5-10%) and sporadic (SALS, 90-95%) forms [2]. Typical age-of-onset is 58-63 years and 47-52 years for SALS and FALS, respectively, with both being phenotypically similar. Unfortunately, the current FDA-approved treatments for ALS including Rilutek (riluzole, a glutamate antagonist) [1] and Radicava (edaravone, a free radical scavenger) [4] only provide limited extension of life; the former acts by attenuating excessive motor neuron firing and the latter works by decreasing oxidative stress.
For FALS, genetic interventions focused on knocking down key genes such as mutant form of super oxide dismutase 1 (SOD1) [5], however, due to the lesser-known etiology of SALS, a successful therapy for ALS will most likely require combinatorial neuroprotective interventions. What is known about ALS, including FALS and SALS, is that they are highly complex, heterogeneous neuropathological conditions resulting from motor neuronal loss due to a highly neurotoxic environment [6][7][8][9][10]. Therefore, interventions that provide neuroprotection may preserve motor neuron survival and neuromuscular function even in the presence of known monogenic mutations or unknown sporadic etiology.
Previously we showed that neuron-targeting of the membrane/lipid raft (MLR) protein caveolin-1 (using a synapsin-driven Cav-1 engineered construct termed SynCav1) promotes hippocampal synaptic and neuroplasticity [11], significantly improves learning and memory in aged mice, and preserves learning and memory in an Alzheimer's disease (AD) mouse model independent of reducing amyloid plaques [12]. Moreover, when crossing our SynCav1 transgenic mouse (TG) with the hSOD1 G93A mouse model of ALS, we observed preserved body weight (BW), longer survival, better running wheel (RW) performance, preserved spinal cord α (alpha) motor neurons, and more MLR-associated neurotrophin receptors (NTRs) in the spinal cord. In terms of human clinical relevance, a seminal finding from the United Kingdom (University of Sheffield) revealed decreased Cav-1 protein expression and disrupted MLRs in samples from sporadic human ALS cases due to mutations in the Cav-1 DNA enhancer region, suggestive of a novel ALS-associated risk variant [13].
One of the major challenges of treating neurodegenerative diseases, including ALS and AD, is to find the most effective manner by which to deliver vectors while minimizing the off-target effect. Although some have demonstrated penetration into the central nervous system and spinal cord using intrathecal or intraventricular delivery using high titers of genetic material, which unfortunately limits the effectiveness and safety of using AAV9-based vectors to treat afflicted spinal cord and brain tissue in ALS [14][15][16][17]. To achieve broader tissue distribution with greater efficacy, the current study utilized the novel spinal subpial (SP) vector delivery route developed by the Marsala laboratory [14].
Over the past few years, the predominant approach to treat ALS is to target known monogenic causes of FALS [15,[18][19][20][21][22][23]. Instead of targeting the genes or proteins attributed to the gain of toxicity, the present study tested the hypothesis that subpial delivery of neuroprotective AAV9-SynCav1 to the spinal cord of hSOD1 G93A mice may mitigate disease progression, protect motor neurons, maintain neuromuscular junctions (NMJs), and extend survival, independent of targeting mutant hSOD1 G93A protein. We now show for the first time that a one-time subpial delivery of AAV9-SynCav1 to hSOD1 G93A mice at lumbar section significantly extends longevity and preserves BW in both male and female mice, preserves lumbar motor neuron morphology and NMJ morphology in the lower limb muscle, and maintains better RW performance independent of affecting expression of mutant hSOD G93A protein. This study further extends the neuroprotective and therapeutic potential of SynCav1 gene therapy to another distinct neurodegenerative condition (i.e., ALS), irrespective of disease etiology, and thus SynCav1 has the potential to treat a variety of neurodegenerative conditions attributed to either familial or sporadic forms. Furthermore, SynCav1 gene therapy may work synergistically in combination with other interventions (e.g., antisense oligonucleotides, antibodies, small molecules) that specifically target monogenic familial forms of ALS and other neurodegenerative conditions.

Animal
Human mutant superoxide dismutase-1 (hSOD1 G93A )-transgenic (Tg) and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and bred in-house. Wild type (WT) littermates were used as control. All animal protocols were approved by the Veterans Administration San Diego Healthcare System Institutional Animal Care and Use Committee (#A20-030). Mice were reared (3-5/cage) with free access to food and water. WT, and hSOD1 G93A mice were allocated to 4 groups randomly: WT, hSOD1 G93A naïve (no surgery), hSOD1 G93A + control (subpial surgery with same volume of AAV9-SynNull or saline), and hSOD1 G93A + SynCav1 to receive subpial lumbar vector injections. Spinal cord tissue was processed for biochemistry, histopathology, and immunofluorescence at the indicated time points.
Sprague-Dawley rats were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) in the Marsala lab and approved by UCSD. Transgenic rats expressing ALS-linked human SOD1 mutant protein (hSOD1 G93A ) exhibit an ALS-like neurodegenerative phenotype characterized by diminished body weight from muscle atrophy, weakness/paralysis, fasciculations, and early death, along with reductions in functional cortical alpha motor neurons and ventral horn alpha motor neurons that can be observed histologically [24]. The hSOD1 G93A transgenic male rats (Marsala lab, UCSD) were bred with WT female rats to produce WT and hSOD1 G93A positive male rats. hSOD1 G93A Genotyping hSOD1 G93A mice and rats were confirmed by genomic DNA extraction and PCR using the Qiagen DNeasy Blood and Tissue Kit (69504; Qiagen, Valencia, CA, USA). PCR was performed for the hSOD1 genes by using the following protocol: denaturation at 94 °C for 2 min, followed by 10 cycles at 94 °C for 20 s, 65 °C for 15 s (-0.5 per cycle decrease), and 68 °C for 10 s, then followed by 28 cycles at 94 °C for 15 s, 60 °C for 15 s, 72 °C for 10 s, then 72 °C for 2 min and hold at 10 °C. All primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). hSOD1G93A-positive mice were confirmed using the following primers: AGC ATC ATC C (oIMR7339)]. These primers were used to detect a product size of 800 for mouse endogenous SOD1 and 600 base pair (bp) for human SOD1 as previously described [25]. Transgenic rats were genotyped by PCR amplification for the hSOD1 G93A . PCR products were separated on a 1% agarose gel (35 min at 135 volts).

Viral vectors and SynCav1 Construct
A self-complementary AAV (scAAV) construct expressing neuron specific synapsin (Syn) promoter with the Cav-1 cDNA was generated at the UCSD Viral Vector core as previously described [12]. To link the neuron specific synapsin (Syn) promoter with the Cav-1 cDNA, an XbaI-SalI DNA fragment containing the Syn promoter was inserted into the NheI-SalI sites of pEGFP-N1 (Clontech). The resulting plasmid was designated pSyn-EGFP. A 537 bp Cav-1 cDNA was isolated from the pCRII-TOPO vector (Invitrogen) by digestion with PmeI-NotI and inserted into the SmaI-NotI site of the pSyn-EGFP to generate pSyn-Cav-1, in which the EGFP gene was replaced with Cav-1 cDNA. The Syn-promoter-Cav-1 cassette was isolated from pSyn-Cav-1. The scAAV vector construct, expressing Cav-1 or RFP driven by the Syn promoter, was made by DNA synthesis of the Syn promoter (480 bp) through Cav-1 (537 bp) or enhanced RFP (700 bp) and cloning it into an scAAV backbone plasmid. The scAAV9-SynCav1, scAAV9-SynRFP, or scAAV9-SynNull vectors were produced by transient transfection of HEK293T cells with each vector plasmid, pRep2/Cap9 and pAd-Helper plasmid. Helper virus-free AAV vectors were produced by Polyethylenimine (PEI)-mediated transient co-transfection of HEK293T cells with three plasmids [pVector, pRep2/CapX (X stands for each serotype) and pAd-Helper]. Cells were collected at 72 hrs post-transfection and cell lysates were made by 3x freeze/thaw cycle. AAV vectors in the cell lysates were purified by combination of sucrose-cushion ultracentrifugation and anion-exchange column chromatography. Virus titers were measured by real-time Q-PCR to determine genome copy (g.c.) number in the vector preparations (g.c./ml) as a measure of AAV particles with full genome content. Final virus titers for all three vectors are ~2.0 x10 13 g.c./ml.

Subpial surgery and AAV administration
All surgical procedures were done under sterile conditions and approved by the local IACUCs. Mouse subpial procedure: Lumbar (L1-L3) subpial vector injection in mice was performed as previously described [14]. Briefly, mice were anesthetized with 2-3% isoflurane, the surgical area was shaved and cleaned, and a skin incision was made at the thoracic 12 (T12)-L1 vertebral level. Under the dissecting microscope, a dorsal laminectomy of the thoracic (T13) vertebrae was performed, and the dura overlying the lumbar (L1) spinal segments was cut open using a 31-gauge (G) needle. The pia mater was then punctured using a 36-G pia-penetrating needle, followed by the insertion of a blunt 36-G injection needle containing the scAAV9 vector. Both the pia-penetrating and the subpial injection needles were mounted on fine XYZ manipulator with needle holder (SM-15HE, Narishige, Japan). Rat subpial procedure: Wild type (WT) or hSOD1 G93A Sprague Dawley rats were anesthetized by isoflurane with 2% in room air and placed into a Lab Standard Stereotaxic frame (Stoelting, Wood Dale, IL). Rats were elevated 3 cm by placing on a plastic box. A homeothermic heating blanket which was set at 37 °C with feedback from a rectal thermometer (Harvard Apparatus, Holliston, MA) was used to maintain body temperature of rats during the procedure. Rats were then placed in Spine Adaptors (Stoelting, Wood Dale, IL) and a C4-5 laminectomy was performed to expose the dorsal surface of spinal segment C4-5 using an air-powered dental drill and binocular microscope. The dura and pia mater were carefully incised using a 27-G needle and a polyethylene tubing catheter, of which its size was 0.008 ID x 0.02 OD, (SAI infusion technologies, Lake Villa, IL) was inserted into the subpia-space. Injections were made using 100-μL Hamilton Gas Tight syringe (Hamilton, Reno, NV). Mice received a total of 10 μl to the lumbar spinal cord and rats received a total of 20 μl of AAV9-SynCav1 (~2.0 x 10 13 genome copies (g.c.)/ml) vector to the cervical spinal cord by subpial delivery (5 μl bilateral to the lumbar spinal cord, 10 μl total over 600 s for mice; 10 μl bilateral to the cervical spinal cord, 20 μl total over 600 s for rats) using a 50 μl Hamilton syringe and digital infusion pump (Microinjector MINJ-PD, Tritech Research). After vector delivery, the muscle and skin were closed using 7/0 Prolene and suture clips. Before recovery from anesthesia, the animals received subcutaneous fluids, antibiotics (cefazolin sodium 50 mg/kg) and the initial dose of pain medication (slow release Buprenex 0.05 mg/kg). Control group received laminectomy and subpial injection of or equal volume of either AAV9-SynNull or sterilized saline.

Assessment of disease onset and survival
Disease onset and late disease were retrospectively defined as the first sign of weight loss from peak body weight and 10% weight decline from peak body weight, respectively [18,26,27]. Survival analyses were performed using Kaplan-Meier analyses as previously described [25]. Survival endpoint is defined by the inability of the mouse to adjust itself within 30 seconds (s) when placed on its back, a humane endpoint that guarantees that animals are euthanized before they are unable to eat or drink.

Voluntary running wheel (RW)
At 8 and 16-week-age, mice were housed individually in plastic cages measuring 40 x 40 x 35 cm (length x width x height) with a 11.5 cm diameter running wheel mounted on the cage side (SuperFlex Open Field, Omnitech Electronics, Inc., Ohio, USA). The mice had free access to food and water ad libitum and were checked daily for health and wellness. The mice had open access to running wheels under conditions of a 12 h light/dark cycle (lights off from 6 AM to 6 PM). The duration of each experiment was 36 h including 2 dark cycles. The mice will be placed in the cages at least 24h prior to data collection. A sensory beam, connected to a desktop computer via a software (Fusion v6.5 r9999 SuperFlex Edition, Omnitech Electronics, Inc., Ohio, USA), was used to record wheel rotations. Running velocity (m/min), and total distance (m) were used for analysis.

Motor Evoked Potential (MEP)
Rats were anesthetized by intramuscular injection of ketamine (100 mg/kg) for motor-evoked potentials (MEP) recordings in the branchial or gastrocnemius muscles. Two 30-G stimulating electrodes were placed subcutaneously overlying the left and right motor cortex. In all rats, MEP of upper forelimb (brachial) and lower limb (gastrocnemius) were recorded from the muscles elicited by transcranial electrical stimulation with a pulse duration of 200 μs at 15 mA. Recording electrodes were connected to an active head stage (Warner Instruments, Hamden, CT), and the recorded signal is amplified using DP-311 differential amplifier (Warner Instruments, Hamden, CT). The amplified signal was acquired by the PowerLab 8/30 data acquisition system (ADInstruments, Australia) at sampling frequency of 20 kHz, digitized and stored using LabChart 7 (ADInstruments, Australia) in PC for analysis.

Grip Strength
Cohorts of age-matched transgenic rats and littermate controls were tested for loss of forelimb grip strength by using a grip strength meter (AMETEK, Inc., Berwyn, PA) at 8 wk (before subpial-injection) and weekly from 10 to 20 wk. Rats were held in front of a horizontal bar, such that only the forelimb paws were able to grasp the bar. The rats were gently pulled back with steady force until both paws released the bar. Each rat was given 6 attempts and the mean grip strength was recorded for analysis.

Statistical analysis
Sample size is indicated under each section of the Methods and in the figure legends. Before determining statistical significance, data were checked for normal distribution. Data were analyzed by Student's t test, one-way analysis of variance (ANOVA) or 2-way ANOVA followed by Fisher's LSD, Tukey's, , or Bonferroni's multiple Šídák's comparisons post hoc analyses as appropriate using Prism 7 software (GraphPad, La Jolla, CA, USA). The Kaplan-Meier survival curve was analyzed using a log-rank test. Mean and median survival were analyzed by an unpaired Student's t-test. Significance was assumed when P < 0.05. All experimental groups were blinded, and code was broken only for analysis. For correlation analysis, a simple linear regression with 95% confidence intervals was performed.

AAV9-SynCav1 delivery to the lumbar spinal cord increases Cav-1 protein expression in the lumbar and thoracic cord
Previous work from other groups have reported that the occurrence of symptomatic onset varies from 8 to 12 weeks (wk) [30] in the hSOD1 G93A mice using different methods and parameters [31,32]. For example, motor deficits and motor unit loss in lower limb muscle due to selective preferential vulnerability of large, fast motor units, innervated by large motor neurons was observed in 8 wk hSOD1 G93A mice, several weeks prior to the standard measurable onset of overt clinical symptoms in this mouse model [33][34][35]. To achieve effective AAV9 transduction of spinal cord motor neurons and peak expression of the Cav-1 transgene (which takes approximately 3-4 wk post AAV delivery), we chose to deliver AAV9-SynCav1 at week 8 via subpial delivery, a highly effective route of administration used to accomplish effective strong transgene expression in the mammalian spinal cord [11,12]. As shown in Figure 1, lumbar subpial delivery of AAV9-SynRFP resulted in a strong expression of RFP in the lower thoracic up to lumbar (T11 to L5), with minimal expression detected in upper thoracic and cervical regions. Our previous study demonstrated that SynCav1+/ hSOD1 G93A + transgenic mice exhibit slowing of disease progression, we therefore initially performed a dose-dependent study for subpial AAV9-SynCav1 delivery to determine the optimal dose necessary to achieve Cav-1 protein expression in the spinal cord similar to that measured in SynCav1+ transgenic mice [25]. As shown in Figure S1, the optimal dose of AAV9-SynCav1 to achieve 4-fold Cav-1 overexpression in the thoracic and lumbar spinal cord was 2 x 10 11 total g.c. (10 μl of 2 x 10 13 g.c./ml). This dose was used in all the following studies.

AAV9-SynCav1 significantly delays BW loss and disease onset, and extends survival in hSOD1 G93A mice
We next assessed whether subpial delivery of AAV9-SynCav1 could provide therapeutic benefit to the hSOD1 G93A mouse model of ALS. As shown in Figure 2A and 2B, compared to hSOD1 G93A + control, hSOD1 G93A + AAV9-SynCav1 mice showed significantly delayed BW loss in both sexes. As shown in Figure 2C and 2D, average disease onset of hSOD1 G93A + AAV9-SynCav1 mice was 18 wk for male (vs 15 wk hSOD1 G93A + control male) and female (vs 16 wk hSOD1 G93A + control female), an approximately 15% delay compared to the control group. In terms of disease progression, late disease onset (i.e., 10% decrease from peak BW) [26] was measured at 20 wk for both male and female hSOD1 G93A + control mice, while AAV9-SynCav1-treated hSOD1 G93A female mice exhibited delayed late disease onset until 23 wk (~11% BW decline); AAV9-SynCav1-treated hSOD1 G93A male mice only exhibited ~4% BW decline at 22 wk (end of body weight record due to loss of multiple objects in the hSOD1 G93A + control groups).

Subpial AAV9-SynCav1 delivery preserves Cav-1 localization to lumbar spinal cord MN plasmalemmal MLRs in hSOD1 G93A mice
As shown in Figure 3A-3B, IB assays of lumbar spinal cord tissue homogenates from terminal stage 23 wk hSOD1 G93A mice (terminal stage as defined by 162 d median survival in Figure 2C and 2D for non-treated hSOD1 G93A mice) showed elevated B8H10 (mutant and WT human SOD protein) and decreased Groups: hSOD1 G93A naïve (no surgery), hSOD1 G93A + control and hSOD1G93A + SynCav1 mice. BW was expressed as mean ± SEM, Two-way ANOVA with Šídák's multiple comparisons test was used for A and B. One-way ANOVA with Dunnett's multiple comparison test was used for C and D (all compared to hSOD1 G93A + control group). Kaplan-Meier Curve was expressed as median days (d) survival and analyzed using a Mantel-Cox Log-rank test (***p < 0.005, ****p < 0.0001).
Cav-1 expression. IB of MLR fractions revealed that Cav-1 was predominantly localized to MLR fractions 4-5 ( Figure 3C) in normal WT mice, which is consistent with previously published work in brain and spinal cord tissue [11,25,29]. However, Cav-1 expression decreased in MLR fractions from the lumbar spinal cords of 23 wk (end stage) hSOD1 G93A mice. Subpial AAV9-SynCav1 delivery to 8 wk hSOD1 G93A mice significantly increased Cav-1 expression and subcellular localization of Cav-1 to MLR fractions, with no effect on expression of mutant SOD protein (B8H10). One limitation to IB assays is that when performed on whole spinal cord (or brain) tissue, one is incapable of distinguishing cell-specific protein changes (i.e., neuronal versus non-neuronal cells). To better elucidate the decreased Cav-1 expression as measured by IB, we additionally performed IF on lumbar spinal cord tissue from 10 wk (early symptomatic) and 23 wk (end stage) using neuronal marker NeuN and Cav-1 ( Figure 3D-3E). While Cav-1 expression exhibited a polarized plasma membrane localization in NeuN-positive large MN cell bodies at the ventral horn of 10 wk hSOD1 G93A mice, a distribution pattern similar to WT mice ( Figure 3E, red arrow heads); lumbar sections from 23 wk hSOD1 G93A mice confirmed that the decreased Cav-1 expression specifically occurred in ventral horn degenerated MN.

AAV9-SynCav1 preserves alpha-motor neuron morphology in the spinal cord lumbar ventral horn and NMJ morphology in the gastrocnemius muscle of hSOD1 G93A mice
We next assayed for lower motor neuron (MN) cell counts and morphology in the lumbar ventral horn region (L3-L5) after subpial SynCav1 delivery ( Figure 5A). As shown in Figure 5B-5E, the total number and cell body (i.e., soma) area of ChAT+ MNs were significantly decreased in both 19 wk male and female hSOD1 G93A naïve mice compared to WT mice, consistent with previous work that showed ALS pathology preferentially affects larger alpha motor neurons innervating faster muscle fibers [36]. In contrast, SynCav1 maintained MN numbers and normal morphology and prevented loss of spinal lumbar ventral horn MNs compared to control hSOD1 G93A mice. Further analysis confirmed that the decreased Cav-1 in the hSOD1 G93A naïve spinal cord is mostly due to the downregulated Cav-1 in the ChAT+ MNs, while SynCav1 preserved Cav-1 expression of the ChAT+ MNs compared to hSOD1 G93A + control mice ( Figure 5F-5G, ****p < 0.0001, Student's t-test, n = 3-8 mice/group). Noticeably, simple linear regression correlation analysis revealed a significant positive correlation between Cav-1 expression and ChAT positive MN area ( Figure 5H-5I, p < 0.0001, R 2 = 0.68). Together, these results indicate that SynCav1 gene delivery affords significant neuroprotection in hSOD1 G93A spinal cord MNs in both Sexes. was performed for B and C = 3-5 mice/group *p 0.05, ****p 0.0001). Student's t-test (n ; < < Finally, because we observed better motor function and preservation of MNs at lower lumbar level with SynCav1 delivery, we analyzed NMJ morphology in the gastrocnemius muscle, one of the larger lower limb muscles receiving synaptic innervation from L5-S1 spinal cord MN axons ( Figure  6A). At 19 wk, mice from both hSOD1 G93A naïve and hSOD1 G93A + control exhibited a significant decrease in NMJ occupancy ( Figure 6B-6C, male, ****p < 0.0001, n = 3-5 mice/group; female, ****p < 0.0001, n = 3-5 mice/group; Student's t-test), with only 30% and 20% of the NMJs occupancy measured in naïve and control hSOD1 G93A mice respectively, indicating severe denervation of the GA muscle [37]. Noticeably, NMJ occupancy in hSOD1 G93A + control mice were significantly lower compared to naïve hSOD1 G93A mice, suggesting an effect from subpial surgery alone on NMJ integrity. In contrast, the NMJ occupancy of synaptophysin in the hSOD1 G93A + SynCav1 mice was significantly greater in both sexes (****p < 0.0001, n = 3-5 mice/group; Student's t-test) compared to hSOD1 G93A + controls, results that in line with the greater RW performance observed in the hSOD1 G93A + SynCav1 group.

Discussion
The present study tested a neuroprotective, non-monogenic targeting gene delivery in the hSOD1 G93A mouse model of ALS. These results show that subpial delivery of AAV9-SynCav1 delays disease onset and early disease progression, preserves ventral horn spinal motor neurons in a familial mouse model of ALS, independent of reducing the toxic monogenic protein engineered to cause the ALS-like neuropathology. Delivering genes that augment neuroprotective proteins may mitigate spinal motor neurodegeneration, prolong survival in individuals afflicted with ALS. The present study showed AAV9-SynCav1 subpial delivery significantly delays disease onset and extends survival in both male and female hSOD1 G93A mice, results that are comparable to previous studies that used ASOs to target mutant monogenic hSOD1 in hSOD1 G93A rodents [26]. Importantly, SynCav1 gene therapy proved efficacious in both sexes, independent of affecting expression of mutant hSOD1 protein. This latter finding suggests that SynCav1 possesses the therapeutic potential to combat other forms of FALS (e.g., SOD, FUS, C9orf72, TDP-43) [6-8, 38, 39] as well as SALS attributed to unknown etiology. Additional work is needed to test whether SynCav1 can work synergistically with other biologics or small molecules designed to knockdown known monogenic links to FALS.
A limitation of the current study is that the extended survival achieved by exogenous vector delivery is slightly lower (10% extended survival) than that observed when crossing SynCav1 transgenic mice with hSOD1 G93A mice (19% extended survival) [25]. This discrepancy between the two studies may be attributed to the following reason: while SynCav1 TG mice overexpress neuronal Cav-1 throughout the whole CNS, the physical and anatomical restrictions of lumbar subpial AAV9-SynCav1 delivery in the small rodent limits broad CNS tissue AAV9 distribution and transduction and subsequent Cav-1 transgene expression. As shown in Figure 1 and Figure S1, Cav-1 transgene expression with SynCav1 predominantly distributed in the lumbar cord (~ 4-fold) and to a less degree in the thoracic cord (~ 2-fold), with no over-expression in the cervical cord after subpial delivery. Failure to target the cervical spinal cord [14], specifically the cervical plexus (C3-C5) which projects motor innervation necessary to regulate diaphragm muscle function, most likely restricted our ability to mitigate respiratory failure in these hSOD1 G93A mice, the most common cause of death from ALS. However, even with this limitation of achieving cervical AAV9 spinal cord distribution/transduction and subsequent Cav-1 transgene expression, the therapeutic efficacy afforded by subpial SynCav1 gene delivery is comparable to approaches targeting the known monogenic cause of the mutant SOD1 neuropathology [40]. For example, a recent study by Powell et al. 2022 [18] delivered their AAV at wk 8 (similar time point in the current study) using CRISPR technology (AAV-RfxCas13d) to reduce mutant hSOD1 by 65% throughout the entire spinal cord (cervical to lumbar) of hSOD1 G93A mice, which demonstrated a similar extension of survival. A combined therapy consisting of SynCav1 with approaches targeting knockdown of monogenic causes of ALS may work in concert to treat ALS more effectively.
On a cellular level, Cav-1 regulates cellular endocytosis and vesicular trafficking [41][42][43], subcellular localization of NTRs and other synaptic signaling components [11,12,29], preserves mitochondrial function in the brain of AD (PSAPP) mice [44] and mitochondrial morphology in the spinal cord of ALS (hSOD1 G93A ) mice [25]. It is well known that mutations in several genes linked to ALS (e.g., Tar DNA binding protein gene (TARDBP) which encodes for TDP-43 [6], fused in sarcoma (FUS) [39], and the C9 open-reading frame 72 genes (C9orf72) [7,8]) impair axonal transport and alter mitochondrial function and dynamics (fission/fusion and trafficking) [9,10,45,46]. Although not investigated in the present study, the neuronal protection and preservation afforded by SynCav1 may in part be through the maintenance/restoration of functional axonal transport of cellular cargoes such as NTR signaling endosomes [47,48] and mitochondria [49]. Using novel in vitro model systems consisting of human motor neurons co-cultured with human myotubes in specialized microfluidic chambers [50][51][52][53] exposed to neurotoxic dipeptide repeats (DPRs) commonly found in FALS [8], will allow the ability to measure axonal transport of organelles and key cellular cargoes [9,49,54,55] to better elucidate the molecular mechanisms underlying the neuroprotective properties of SynCav1.
Preservation of NMJ morphology is essential for preserving motor function and life quality. NMJs are unique synapses that facilitate communication between MNs and the effector muscles. Due to their long axons, MNs are extremely susceptible to retraction during pathological conditions such as ALS, thus resulting in disrupted communication between the MN and effector muscles. Initially, the MNs try to compensate for the retraction by inducing axonal sprouting and collateral innervation. However, with disease progression, this compensation fails and results in final MN death, a neuropathological event known as the "dying back" mechanism [56]. The subsequent NMJ disruption causes significant skeleton muscle atrophy, respiratory insufficiency, and death [57,58]. The present study revealed that AAV9-SynCav1 preserved NMJ integrity and improved running wheel performance in both hSOD1 G93A male and female mice. Could SynCav1 be applied at this MN 'compensatory stage' to prevent or overcome the "dying back" mechanism and maintain functional NMJ synapses? As noted above, the use of specialized in vitro models consisting of human motor neurons forming functional NMJs with human myotubes could optimize the temporal window at which to deliver SynCav1 in the presence of otherwise toxic dipeptide species to overcome this "dying back" cellular event. Another limitation to the current study is the lack of more sensitive neuromuscular measurements of functional NMJ physiology in the upper or lower limbs (e.g., MEPs, grip strength) performed specifically in the hSOD1 G93A mouse ALS model. However, using the larger hSOD1 G93A rat ALS model, subpial cervical spinal cord SynCav1 delivery resulted in significantly better forelimb grip strength and MEPs (greater amplitude and decreased latency) in both upper limb brachial and lower limb gastrocnemius muscles, further evidence of SynCav1's efficacy using two different ALS model species.
The current study delivered SynCav1 at week 8 (pre-symptomatic stage) to the lumbar cord which exhibited similar folds increase as observed in the healthy wild type mice. However, vector dosing needed to achieve efficacious transgene expression levels may depend upon disease stages [59]. If vector is delivered at a later stage, Cav-1 expression in the lumbar cord neuron may fail to achieve the same level of fold increase since the neurodegenerative spinal cord may not hold the capacity to express an exogenous transgene to the same levels as healthy spinal cord, an important pre-clinical finding to consider when formulating human equivalent dosing to achieve clinical efficacy [60][61][62][63]. Interestingly, we measured adverse effects from the subpial surgery on BW at the late stage of disease compared to the naïve hSOD G93A mice, with no effect on disease onset or survival. A possible explanation is that the surgery may have exacerbated back muscle weakness, which in turn could have affected the ability to retrieve food located on top of the cage for late stage hSOD1 G93A mice. This inability to retrieve food would result in a more rapid BW decline compared to naïve hSOD1 G93A mice. The fact that we did not measure any difference in survival, MN morphology or MN count between hSOD1 G93 control and hSOD1 G93 naïve mice, indicate that the adverse effect from subpial surgery on BW is not through affecting MN survival directly. Nevertheless, since the lumbar subpial surgery involves an invasive laminectomy, which is difficult to perform successfully on older, severely symptomatic hSOD1 G93A mice. Exploration of alternative routes of SynCav1 administration (i.e., intrathecal and intracerebroventricular) are worthy of investigation.
In summary, the present study demonstrates that subpial SynCav1 gene delivery to the lumbar spinal cord of hSOD 1G93A mice prolongs survival and maintains body weight in both males and females, preserves alpha motor neurons in the ventral horn with a direct correlation to soma size and Cav-1 expression, and preserves NMJs in the lower limbs, all of which occurred independent of affecting expression of mutant hSOD1. These data suggest that SynCav1 might serve as a novel gene therapy for neurodegenerative conditions in ALS and other forms of CNS disease of unknown etiology. Further studies are needed to determine the effect of SynCav1 on disease progression when delivered at later stages of the disease.